Need to Know

Formation of a biological membrane

Unit 1. Phospholipids (A) are amphipathic molecules with a hydrophilic phosphate head and a hydrophobic tail of fatty acid chains.

In an aqueous environment the hydrophilic heads move to stay in contact with water and the hydrophobic tails move away from the water but toward each other. This forms a hydrophobic interior that is “protected” from water on either side by the hydrophilic heads to organize a phospholipid bilayer (B). 

(C). The 3D effect of being in water: when hydrophobic tails are exposed to water along the edges of the bi-layer, they will move to be in contact with the hydrophobic tails on all sides. This spontaneously creates an enclosed spherical structure. This illustration has a cut-away portion so you can see the interior structure of the bi-layer. In reality, the membrane would be organized into a complete hollow sphere.

Note: Phospholipid bi-layers present provide a biological barrier to gate or slow the passage of things from the inside out and from the outside in. This makes it very useful in being able to concentrate biologically important molecules in a small area to increase the likelihood of molecules coming together to perform biologically important reactions! On the downside, this large zone of hydrophobicity makes it difficult to pass hydrophilic substances from one side to another. Think about the last time you tried to make a vinegarette using oil and vinegar. Life—uh—finds a way to overcome this by leveraging the chemistry of other molecules…think about how you could solve this problem as we explore more about the biochemistry of membranes.

Highlights about Amino Acids

 There is a root structure to amino acids (unhighlighted portions in the figure below).

Think about the different categories of amino acids. These chemical characteristics (non-polar, polar, aromatic, positive and negatively-charged) set the rules by which each one of these building blocks can interact with each other and with the surrounding milieu of molecules. Recall from General Biology that these molecules are chained together with covalent bonds (see the figure below to refresh yourself on how these covalent bonds are formed) to form the primary structure of a protein.

The different characteristics of the R groups of amino acids are critical to understanding how a protein sequence will fold into its secondary and tertiary (and even quaternary) structure and predicting how these structures contribute to function. Take for example, the protein hemoglobin which carries oxygen in the blood. Oxygen is hydrophobic in nature, but it must be carried in an aqueous environment (blood). Given this information and the following diagram, what predictions could you make about the type of amino acids you would expect to find in this molecule? 

Think back to the scenario in the previous section: the problem of getting hydrophilic molecules from the aqueous environment on one side of the membrane through the hydrophilic environment in the middle of the bi-layer membrane. Be prepared to propose a solution to this issue in the next class meeting. Unit 2.Where things meet = point of interest

One core concept that you will find arise again and again in your coursework and beyond is: interfaces are critical regions in cell biology! A prime example of an interface is that of the membrane and the cell cytoplasm, or the membrane and the exterior world of the cell. In eukaryotes, you have cell membranes, membrane-bound organelles, and vesicles creating interfaces that represent the site of interaction, reaction, separation, protection, selection, targeting, etc. In the prokaryotic world of bacteria, there is also the bacterial envelope to examine.

Introducing the bacterial envelope

Most (though not all!) bacteria can be sorted into either gram negative or gram positive based on the chemical structure of their bacterial envelope (see the image below). If you want to know more about the bacteria that don’t fall into these two categories, we recommend looking forward to medical microbiology and pathogenic microbiology courses! For now, let’s focus on these two categories.

Review the provided diagram of the Gram-positive (top) and Gram-negative (bottom) bacterial membrane + envelope. What are some of the key differences you can observe between these two types of structures? What are some commonalities? In both Gram + and Gram – bacteria, we see the presence of peptidoglycan . This is a glycoprotein (sugars + amino acids) (see the diagram below) that consists of chains of sugars (N-acetylglucosamine (abbreviated as either NAG or GlcNAc) and N-acetylmuramic acid (abbreviated as either NAM or MurNAc)) that are bridged together or cross-linked by short peptide bridges. 

This structure forms a molecular netting (kind of like support stockings or a cargo net). Just like with compression stockings, the net-like structure makes the peptidoglycan very resistant to mechanical stress and can be thought of as bacterial shapeware; it confers the shape and the rigidity to the bacterium.

Inside the bacterium there is high internal pressure (as high as in a car tire!) due to the concentration of molecules inside the cell versus the external environment. Think back to what you learned in general chemistry and general biology about the principles of osmosis: water will want to flow across a permeable membrane in the direction of higher solute concentrations to reach a state of solute: solvent balance or equilibrium. Recall, too, that one purpose of cell membranes is to allow the concentration of molecules (solutes) essential to life processes. Taken together, the higher concentration of molecules inside the bacterium promotes diffusion of water from the environment to the inside the microorganism. Result? The bacterium will tend to swell and explode.

It is important to note that the peptidoglycan layer will not restrict the diffusion of water inside the bacterium but will prevent the microorganism from swelling and exploding by conferring a rigid case that will restrict the bacterium swelling. Repeat: peptidoglycan is fully permeable to water.

Now, what happens when you cut or puncture a set of support stockings? In the same manner, if the peptidoglycan is damaged or if it cannot be effectively manufactured when the cell is preparing to divide, what would you predict would happen? Be thinking about the implications and outcomes for class discussion!

One more look at the difference and similarities of the bacterial envelope of Gram + and Gram – bacteria. 

Looking at our same diagram from before, we know that peptidoglycan is a shared feature that is unique to prokaryotes and distinguishes them from eukaryotes like us! But, note that Gram+ bacteria have a much thicker shell of it. Gram+ bacteria also have teichoic acid studding the peptidoglycan like structural pillars of a building. Just like structural pillars, these chemicals reinforce and anchor the peptidoglycan layer. These acids also play a role in metabolism, immune response, and virulence. Overall, though, the Gram + envelope is fairly simple in design when contrasted against Gram – microbes. 

In addition to a peptidoglycan layer (which is quite thin!), Gram - microbes have another component sitting atop the peptidoglycan layer called the outer membrane. Importantly, the role of the outer membrane is the same as the cell membrane: to act as a semipermeability barrier to the diffusion of molecules in and out of the cell. And just as with the cell membrane, the outer membrane is studded with proteins to help facilitate movement of molecules in and out of the cell and carry out other important functions.

If you compare the outer membrane with the cell membrane you will observe some other structural similarities like having a phospholipid layer. But, don’t confuse the outer membrane for the cell membrane! They are separate and distinct structures! A critical distinction in the composition of the cell membrane and outer membrane exists, too. Whereas both inner and outer layers in the cell membrane are made up of phospholipids, the outer membrane has a phospholipid layer on the inner surface, and a unique compound called lipopolysaccharide (or LPS) that makes up the outer surface.

Lipopolysaccharides are very toxic to mammals; however, the bacterial role of the outer membrane is NOT to cause toxicity. Human toxicity and pathogenicity is an unintentional side effect of the process of adaptation, competition and survival.Unit 3.Other structures on the bacterial surface

There are three major types of structures that can be found on the bacterial surface: fimbriae, pili, and flagella (see the image below). Fimbriae are short structures anchored in the outer membrane of a bacterium (would this be a Gram + or Gram – bacterium?) that act like molecular Velcro to help the microbe adhere to surfaces.

Pili are more complex structures both in form and function. These longer extrusions of the cell are also involved in attachment but can be used to transfer DNA from a doner bacterium to a recipient bacterium through a process called conjugation (more on that later). It can also be used by the bacterium to move randomly through a twitching process. Note, too, that unlike fimbriae, the pili are anchored in the inner membrane of the bacterium and pass through the outer membrane through a collar of proteins that allow the assembly to pass through.

One reason why attachment is important to bacteria is that, in the wild, bacteria do not live alone. Just like everything, it lives within an ecosystem and forms communities with other bacteria of its own kind and of different kinds. These communities (called biofilms) offer support, create structures to help trap and concentrate nutrients, but also represent a concentration of population competing for resources over time. This can lead to chemical warfare when the population gets too crowded! It is also a place where bacteria can exchange (or steal!) information in the form of toxins and resistances to toxins that are developed in the biological arms race. Can you guess why this matters to us?

In addition to fimbriae and pili, there is also a more sophisticated structure called the flagellum (see below). The flagellum is an even larger complex of proteins than the pili and is anchored in multiple locations throughout the bacterial cell membrane, peptidoglycan layer, and, if Gram -, the outer membrane. This is a molecular motor structure which requires energy to drive a rotating motion. Note the hook or bend in the flagellum structure in the illustration. This bend provides a kind of corkscrew effect in aqueous environments, allowing a bacterium to push in a (mostly) unidirectional motion. This enables the bacterium to move away from toxic or nutrient-deplete environments or to drive attachment activity in establishing biofilms.

Biofilms and why attachment matters

The image below depicts major functions of a biofilm. A biofilm is a collection of bacteria (usually a mix of different species in the wild) making a community within what is called the extracellular polymeric substance (EPS) or extracellular polymatrix (EPM). Made largely of polysaccharides, this matrix creates a stable, but permeable environment that provides numerous advantages to the local bacterial residents. Biofilms differ from single-species bacterial colonies like you would grow on a petri dish in a research lab in many ways including the genes expressed for metabolism, and compounds for offense and defense (toxins, antibiotics, and antibiotic resistance genes). Take a moment to review the diagram and see the benefits living in an eclectic biofilm community can bring!

Surface structures help bacteria form these communities and the bacteria will go through a cycle of being free-floating to being part of a biofilm and back to being free-moving (usually when the biofilm is under nutrient stress from overcrowding). Just like when people move out of an overcrowded city for the fresher air of the suburbs, bacteria will pack up and move when the going gets tough!

Biofilms and genetic exchange

When bacteria cluster together in biofilms, this becomes a hotbed of exchange, not just of nutrients or trade-off of performing different metabolic tasks for the community, but also of genetic information. Whether through conjugation or through the uptake of genetic information from bacteria that have burst or decomposed in the matrix (transformation), or through the assistance of viruses that target bacteria as their hosts carrying some of their previous host’s DNA to their new victim (transduction) lateral gene transfer is a major means by which bacteria gain new properties. The biofilm provides a concentration of different organisms thus increasing the chances of acquiring new traits! To understand a little bit more about the types of genetic exchange that happen in these spaces, let’s take a quick detour to understand a bit more about bacterial DNA.

Sources of genetic information in a bacteria

In the image below, we see three types of bacterial DNA: the chromosomal DNA, and two types of non-chromosomal DNA (plasmids and transposons). Chromosomal DNA belongs to the cell, exists in one copy, and only replicates when the cell replicates. This contains all of the instructions necessary for the bacteria to function. Non-chromosomal DNA, though, plays by some different rules. Plasmids, for example, can exist in many copies in a single cell and can replicate independently of cell division. These small, circular pieces of DNA can carry a set of genes (often several genes!) that can impart antibiotic resistance, stress-survival properties, toxins…they can even be used by researchers to insert specific instructions to make human-useful things like insulin or vitamins! Plasmids can also carry information that allows a bacterium to directly transfer DNA to another through a process called conjugation (see below).

Transposons, are a different kind of non-chromosomal DNA. Unlike plasmids which are large pieces of DNA that are independent of the bacterial chromosome, transposons are small pieces of DNA that integrate into the bacterial chromosome. These small pieces of DNA also carry instructions that can impart novel properties to the recipient of the gene through the process of mutating the genetic code. They can also spontaneously move around or “jump” from one location in the chromosome to another. Because of this ability to move about, they are often referred to as “jumping genes” or transposable elements.

Let’s take a moment to appreciate why plasmids and transposons matter to humans:

  Unit 4.Bacterial communities and the dynamics of prokaryotic growth

On the right, we have an example of a bacterial growth curve. This graph belongs to a pure culture in nutrient broth (conditions that we see in most research laboratories!). Take a moment to review the features.

Consider 2 key questions for when you meet in class:

1. How would you determine the number of cells in liquid culture in order to generate this curve?

2. How would you describe what is happening in each phase and what are the reasons driving each phase?

Note, too, that we stated the curve above represents a liquid monoculture or a pure culture of just one species and strain of bacteria. This is also a bit of an idealized growth curve. What might happen in mixed cultures or in the messiness of real-world situations like a biofilm?

In the biofilm the growth kinetic is determined by many environmental factors that are irrelevant in the laboratory conditions where they are controlled. In the wild, for example seasonal variations in temperature affect the growth kinetic of micros in the biofilm but not in a laboratory culture where things are kept at a steady temperature (often 37⁰C…why is that? What pitfalls could this result in?).

However, regardless of whether you are examining a biofilm or a monoculture, the availability of nutrients is a common factor that affect both. In biofilms the population and composition of microbes in the community + nutrient availability partially dictates the type of predominant metabolism. During starvation, primary metabolism switches to secondary metabolism and microorganisms start producing antibiotics. Antibiotic-producing microorganisms are resistant to the antibiotic that they produce (those that can’t kill themselves off!) but the heterogenic nature of the biofilm allows elimination of competitor microbes that are susceptible to the antibiotic being produced (see below). DIABOLICAL!

This effect, though, creates a new balance between the cell number and nutrient availability. This adaptation does not occur in a pure culture since all the microorganisms in the pure culture are identical and although antibiotics are produced at the end of the log phase and during the entire stationary phase that does not change the cell number and the culture progress into the death phase. 

Key points to synthesize from this unit:

1. Bacteria need nutrients to grow.

2. Bacteria in cultures/communities produce waste products and secondary metabolites that could prove toxic to the culture (insert social commentary here).

3. Adaptation to counter toxins, metabolize new food sources, produce toxins (antibiotics) can be acquired through lateral gene transfer which can occur by several different routes (know the differences!).  That which does not kill them, makes them stronger!

4. As we invite greater complexity into the system, the growth curve changes to illustrate the interrelationship of growth, metabolism and selective pressures.

My Lecture Notes:

Micro Module 1 Lecture 1-5 is module 1 We will go over on Thursday 

Objectives :

Identify and describe the structure and functions of the cell membrane and bacterial envelope.

Basic understanding:

• Compare and Contrast

- Chemical composition of these structures with eukaryotic cells

-architecture of these structures with eukaryotic cells 

•  describe relationship between structure and basic functions of the cell

  •  (survival, competition, growth, control of influx and efflux of materials)

Advance understanding: 

• relevance and implications of these structures (including their chemical properties) to

  •  with an eye to clinical, industrial, environmental, and agricultural settings.

Explain the relationship between bacterial locomotion and attachment to host cell surfaces through the lens of virulence factors.

Basic understanding: 

• explain the link between structures ( like the bacterial pili and flagellum) and  bacterial “success” strategies (how well it evades predation and poor or noxious environments, finds nutrients, creates communal environments like biofilms)

Advanced understanding: 

• How structures contribute to selection and competition pressures, 

• How structures create competitive or cooperative environments (and be aware of the contribution of these structures play in the “accident” of human pathogenicity and clinical challenges)

  • start linking the nature of monocultures, liquid cultures, and plate cultures grown in labs compared to the behavior and composition of bacterial communities “in the wild.”

Articulate the componentry and contributions of chromosomal and non-chromosomal DNA:

Basic understanding: 

• two types of DNA sources in bacteria and their distinct means of transfer to other bacterial cells. Differences in source, size, and replicative behaviors

  • chromosomal 

  • non-chromosomal (plasmids and transposons)  

Advanced understating: 

• identify conditions that make lateral gene transfer more likely to occur, 

• articulate the potential advantages to a bacterium receiving genes from another organism

• link these gene transfer events to clinical, agricultural, research, and industrial relevance or application.

Obj 4) To understand how microorganisms grow, how their growth is measured quantitatively in the laboratory, and how microbial populations are controlled using physical and chemical methods 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

  Obj 1) Identify and describe the structure and functions of the cell membrane and bacterial envelope(what is outside the cell wall)

Eukaryotes -

• Plants

• Fungi

• Animals

Protist -

• Anything else

Archea - 

• Extremophiles live in extreme conditions 

HO is polar hydrophilic bc oxygen is a bully and increases electronegativity

In the small world we are limited to describing morphology by shape. Shapes have to do with where they live.

KNOW THE ROOTS 

Prokareyotes (bacteria and archea no nucleus) 

 Spherical shape or sort of spherical (coccus means spherical)

Each bacteria is an individual cell

Rod shapes ( bacillus)

Curved rod shapes or squiggly shapes

Some Archea have a post it stamp shape flat rectangle 

Eukaryotes (nucleus)

Lipid Bilayer 

Lipid bilayer 

(in bacteria is similar to the ones in our cells)

An oily layer defines the outside of a cell

Polar head (like water) 

If you’re hydrophobic, you need to surround yourself with hydrophilic environment (like the heads on a phospholipid bylayer)

Phospholipid Bilayer 

THE BUILDING BLOCK OF A CELL MEMBRANE

Saturated fatty acids/lipids = linked by single covalent bonds 

Unsaturated = multiple covalent bonds 

The Cell Membrane of bacteria

• The Cytoplasmic Membrane(no difference than cell membrane or plasmic membrane)

• ((Plasma Membrane (the same as cell membrane just different name))

  • Hydrophilic heads and hydrophobic tails

  • Lipid-soluble molecules can easily get across (molecules like to go down in concentration that requires no energy until it’s evenly spread, if it uses energy to move something against concentration gradient that is active transport!)

    • Phospholipid Bilayer has

      • Embedded proteins that are to transport molecules that are hydrophilic because they have a hard time getting through the hydrophobic inner part which are non lipid molecules or IONS

        • Lipid soluble molecules can get through easy

      • HYDROPHOBIC = PHATTY ACIDS which is the MIDDLE REGION , non polar covalent bonds because a long chain of carbon and hydrogens 

      • HYDROPHYLLIC = glycerol +phosphate and another functional group like sugars, choline etc.)the blue part is hydrophilic (idea of electronegativity)

    • Some bacteria species are strengthened by HOPANOIDS these are sterol-like molecules (act like cholesterol in eukaryotic cells)

    • Proteins 

      • Integral membrane proteins are significantly attached. They got INTO membrane 

      • Peripheral membranes are loosely attached just on the outside.

    • Quiz Q: what would adding the haponoids do to the membrane?

      • They might change the way the molecules move. The haponoids slow the oil movement around. Because each head on either side is transient they move they don’t stay with their buddy on the other side. So the haponoids add rigidity which changes the characteristics of a bacteria. When oil is cold it turns harder into a fatty thing which means the nerves in your fingers can’t work, the fat will change how the cell membranes move. 

-Archeal Membranes

• The carbon hydrogen chain(fatty acid tails) look different. Its just double carbon (its called isoprene side chain)

• Ether bond in archea instead of ester bond 

• Glycerol in archea is L glycerol and in bacteria/eukaryotes is D glycerol (L and D are mirror images of each other)

• Bacterial Cell Walls: Peptidoglycan

• Lipopolysaccharide (LPS): The Outer Membrane

• Archaeal Cell Walls

Bacterial Envelope: Gram + and Gram -

Gram + :

• Thicker peptidoglycan shell

• Stain purple

• Will often have a continuous row of Gly 

• Teichoic acid(Glycopolymers) going through the peptidoglycan layer like pillars

  • They are negatively charged over all

  • Sometimes irs Lipoteichoic acid 

    • Just means it has a hydrophobic end 

  • These strengthen structure

  • Role in metabolism, immune response, and virulence

  • Are negatively charged

  • Required for virulence and adhesion

• Gram-positive advantage → mechanical strength and environmental resilience

Gram - :

• Thinner peptidoglycan shell

• Stain pink

• Have a link from DAP to d-ALA

• Have an outer membrane

  • Both cell membrane and outer membrane are made up of phospholipids

  • Outer membrane (difference between cell membrane and outer membrane)

    • Composes most of the cell wall

    • Has an inner phospholipid layer on inner surface

    • lipopolysaccharide (or LPS or lipid sugars) on the outer surface

      • Endotoxin: Lipid A ( the toxic part of LPS)

      • Barrier against anti biotics / harmful agents

• Harder for molecules to move in an out!!!

  • Not just because peptydoglycan 

  • Its the combo of hydrophilic and hydrophobic 

• Gram-negative advantage → chemical defense (outer membrane)
  
  

Similarities between Gram - and Gram +(Bacteria normally falls into this) :

-Peptidoglycan(this is what stains)

- This is glycoprotein (sugar and amino acids)

- its sugars going left to write with links going up and down that are peptide bridges

- They both have proteins on the cell membrane (Gram + ) or outer membrane (Gram -) to help facilitate movement of molecules

Quiz Qs:

What happens if the “supportive stocking” get cut or punctured?

The bacterial cell will explode. Peptidoglycan is fully permeable in water.

Cell Wall: Peptidoglycon (dont need to know the chemical structures but i need to know the names, unique to bacteria)

• Composed of alternating modified glucose to form a sheets on top of sheets on top of sheets that can move on top of each other sliding BUTTT to link them together with amino acids

  • N-acetylglucosamine 

  • N-acetylmuramic acid

• Can be destroyed by 

  • Lysozyme (an Enzyme)

  • It’s job is to break down cell wall of bacteria you consume (its in our saliva)

    • They are very specific though to certain molecules in this case Beta 1- 4  linkage but it would not recognize a Beta 1-3 linkage or bond

Key Concepts with Bacterial Envelope 

1. Bacteria envelopes have a different composition in Gram positive and negative microorganisms.

   1. Gram positive: Thick peptidoglycan

   2. Gram negative: Thin peptidoglycan and an outer membrane

2. The bacterial envelope does not include the cell membrane.

3. Consider the implications (pros and cons) between G+ and G- bacterial envelopes

   1. Environmental survival 

   2. Transportation of molecules in and out of the cell

   3. Clinical implications (Abx resistance, etc.)

Big Picture Summary (Exam-Ready)

• Cell membrane: 

  • selective barrier, energy generation, transport

• Cell wall: 

  • shape, rigidity, osmotic protection

  • 
    
    

• Gram differences: 

  • thickness of peptidoglycan and presence of outer membrane

• Structure directly supports function in survival, growth, and resistance

Basic understanding:

• Compare and Contrast

- Chemical composition of these structures with eukaryotic cells

- Architecture of these structures with eukaryotic cells 

•  describe relationship between structure and basic functions of the cell

  •  (survival, competition, growth, control of influx and efflux of materials)

Advance understanding: 

• relevance and implications of these structures (including their chemical properties) to

  •  with an eye to clinical, industrial, environmental, and agricultural settings.

1. C

2. C

3. E

4. D

5. A

MINOR DIVE

How would a bacterium’s survival change if it’s cell membrane suddenly became too rigid or too leaky?

I think they would die because the cell membrane controls organelles and molecules . It would not be able to keep in what it is supposed to or keep out what it is supposed to. It could possibly de-hydrate or get too much water.

Cell Surface Structure (Lec2)

Capsules and Slime Layers

1. Not cell wall bc provides less structure 

2. Both Polysaccharide (May be thick or thin, basically sugar)

   1. Assist in attachment ot surfaces

   2. Maintains biofilms

      1. Where you get lots of organisms sticking together

   3. Virulence Factors : Protect against phagocytosis!!!!! (virulence factors means help attach to host and get in body)

      1. Phagocytosis is an immune system cell that engulfed entire bacterial cells to kill it.

         1. They do this by detecting things outside the cell wall but with the layer it protects it from sensing it

3. Capsules 

   1. Tightly attached

4. Slime Layers

   1. Loosely attached 

Fimbriae and Pili

1. Fimbrae

   1. Protein Structure 

   2. Short and many

      1. Enables sticking to surfaces

2. Pili

   1. Few and long

   2. Protein Structure 

      1. Conjugative/sex pili facilitate genetic exchange 

3. Type IV Pili

   1. Adhere to host tissue

   2. Support twitching Motility 

Cell Inclusions 

1. Reduces osmotic stress (stress by change in solute concentration around it)

2. Internal Storage Structures

   1. Energy Reserves

   2. Carbon Reserves

   3. Stores other essential molecules for metabolic function

Gas Vesicles 

1. Allows buoyancy 

   1. Gas filled conical shaped structures

   2. For photosynthetic bacteria 

 Endospores

1. Designed to survive extremes bc of dry DNA and layers of protein 

2. Present in only some Gram + Bacteria (Bacillus and Clostridium)

3. Survival structures 

   1. Help endure nutrient deprivation 

   2. Dispersal by wind water or animal gut

4. Growth Steps

   1. Activation

      1. brief sub-lethal heat

   2. Germination

      1. loss of resistance and refractivity

   3. Outgrowth

      1. water uptake, RNA/DNA/protein synthesis

5. Dipicolinic Acid 

   1. The core contains SASP( small acid-soluble spore proteins)

   2. Dehydrators the DNA in developing endospore 

      1. This is to protect it from the radicals when water gets hits with high radiation 

      2. Inserts between dna bases for stability against heat denaturation(forming radicals)

Cell Locomotion

Flagella and Archella 

1. Assist in Swimming Motility

2. Flagella (bacteria)

   1. Structures, keeps inner and outer membrane of gram - bacteria together

   2. Long and thin

   3. Move by rotation

   4. Powered by proton motive force

      1. Proton Turbine

3. Archealla (archea)

   1. ATP hydrolysis provides force for archaeal rotation

   2. Move by rotation

4. Gliding Motility 

   1. Movement requires to be on solid surface

Obj 2) Explain the relationship between bacterial locomotion and attachment to host cell surfaces through the lens of virulence factors.

Cell Locomotion

Flagella and Archella 

5. Assist in Swimming Motility

6. Flagella (bacteria)

   1. Structures, keeps inner and outer membrane of gram - bacteria together

   2. Long and thin

   3. Move by rotation

   4. Powered by proton motive force

      1. A motor in cell membrane

      2. Mot protein is battery protons come into it (h+)

         1. The H+ ions are higher outside the cell wall then inside so they go down the concentration gradient and go inside the MOT protein

      4. Proton Turbine

   5. Peritrichous (all over the place glagella) vs Polar Flagella 

      1. Peritrichous moves counter clockwise to move and then clock wish which open sup the multiple flagella to stop

      2. Polar Flagella move clock wise or counter clockwise to move opposite direction

7. Archealla (archea)

   1. ATP hydrolysis provides force for archaeal rotation

   2. Move by rotation

8. Gliding Motility 

   1. Movement requires to be on solid surface

   2. ​​•Mechanisms:

      1. •excretion of polysaccharide slime (cyanobacteria aka photosynthesiers)

      2. •type IV (4) pili/twitching motility

      3. •gliding-specific proteins (adhesion complexes or other specialized proteins)

9. Taxes

   1. Movement towards stimuli or away from repellent 

      1. •chemotaxis: response to chemicals

      2. •phototaxis: response to light

      3. •aerotaxis: response to oxygen

      4. •osmotaxis: response to ionic strength

      5. •hydrotaxis: response to water

1. D (I put E )

2. B

3. B

4. E

5. E

Obj 3) Articulate the componentry and contributions of chromosomal and non-chromosomal DNA:

Basic understanding: 

• two types of DNA sources in bacteria and their distinct means of transfer to other bacterial cells. Differences in source, size, and replicative behaviors

  • chromosomal 

  • non-chromosomal (plasmids and transposons)  

Advanced understating: 

• identify conditions that make lateral gene transfer more likely to occur, 

• articulate the potential advantages to a bacterium receiving genes from another organism

• link these gene transfer events to clinical, agricultural, research, and industrial relevance or application.

Genetic Elements : Chromosomes and Plasmids

Chromosomes -

• Maine genetic carrier in prokaryotes (DNA)!!!

• Plasmids(circular pieces of DNA but the difference is size.)

• Transposable Elements

• Proteins!!

• Millions of base pairs longs

• All important genes basics

  Bacterial Chromosomes

• 1 Single circular chromosomes 

• This is cells instructions manual

Plasmids 

• Upgrades

• Smaller circular pieces of DNA 

• Thousands of base pairs long

• Separate form chromosome

• Replicate on their own!!!! 

  • Not required for survival 

• Purpose/Why they matter

  • Carry antibiotic resistance genes(R Plasmids)

  • Virulence factors 

  • Metabolism

  • Bacteriocins (can be encoded)

    • Proteins that kill or inhibit close related species or different strains of the same species

    • Stressful environment where competition arises

R Plasmids 

• Antibiotic resistance gene 

Non-chromosomal Bacterial DNA - Transposons (Jumping Genes)

Transposons

• Different from plasmids 

• Can insert into chromosomes and plasmids

  • Replicate when chromosomes replicate

• Creates Mutations!

• Spread antibiotic resistance 

• Spread virulence factors

Three Ways Bacteria Share DNA - Mechanisms of Lateral/Horizontal Gene Flow (Sharing Anti Biotic Resistant Genes)

Transformation

• DNA is released by one cell then taken by another 

  • Cells die and DNA is free to be able to take up

• To take up: cells have to be competent (a lot of bacteria is non-competent)

  • Competent -

    • Competent cells are cells that can take up DNA and be transformed

Transduction

• Viruses that infect bacteria sometimes randomly get fragments of DNA and carry to other cells

• Done with bacteriophages (phages)

• Generalized Transduction

  • DNA form any part of host is put inside virion

  • This DNA cannot replicate independently and will be lost without recombination

  • Not very efficient or common 

• Specialized Transduction

  • DNA from a specific part of host’s chromosome is put in virion

  • May integrate during lysogeny 

  • Or homologous recombination can occur

  • Very efficient 

Conjugation

• Transfer by cell to cell contact through pilus

• Donor Cell (F+)

  • Has conjugative plasmid

  • Copies DNA keeps one copy gives another 

  • Tra gene facilitates transfer of DNA between cells

• Recipient Cell(F-)

  • Does not have conjugative plasmids

Hfr Cells(These are cells containing an integrated F plasmid F+)

• Cell becomes Hfr (High frequency recombination)

• Can transfer chromosomal genes

• Leads to more recombination

• Plasmid recombines with chromosome of another cell 

Insertion Sequences

• Simplest transposon 

• Small piece of DNA able to jump to different locations in DNA

• Can insert in chromosome, plasmid, and transposon

• An insertion sequence is a small jumping DNA piece that carries only the enzyme needed to move itself and can cause mutations when it inserts into new DNA locations.

• No antibiotic resistance genes 

1.D

2.D

3.E

4.A

5.C

Obj 4) To understand how microorganisms grow, how their growth is measured quantitatively in the laboratory, and how microbial populations are controlled using physical and chemical methods 

1. Mechanisms of Microbial Growth

• Growth 

  • Increase # of cells, cell division

• Binary Fission 

  • Cell division after enlargement of a cell to twice its minimum size into two daughter cells

• Septum

  • Partition or wall between dividing cells 

• Generation time 

  • Time required for cells to double in number 

    • Depends on

      • Nutrition 

      • Genetics

      • Temperature 

• Biofilms

  • Prevent harmful chemicals from entering, protists from grazing, and washing away of cells PROTECTION

  • Planktonic Growth

    • Growth as suspension

  • Sessile Growth 

    • Attached to surface 

  • Matrix 

    • This is the biofilm and more friends come along and get stuck in the matric and make more. 

    • Transformation is efficient in here because dna is held in matrix

    • The matrix of a biofilm is primarily composed of extracellular polymeric substances (EPS), which are also often referred to as the "slime" or "glue" of the microbial community.

      • Polysaccharides and proteins.

  • Microbial Mats 

    • Multilayered sheets with different organisms in each layer 

      • Hot springs 

      • Intertidal Regions 

2. Quantitative Aspects of Growth

• Exponential Growth 

  • Population double within a specific time

• Generation time 

  • g= t/n

    • T = duration of exponential growth days 

    • N = number of generations during the period of exponential growth 

    • G = generation time

• Instantaneous Growth Rate Constant 

  • K = rate of growth at any instant 

  • Inversely proportional to generation time 

3. The Microbial Growth Cycle (Batch Culture)

• Batch Culture 

  • Closed system with fixed volume microbial culture (dont add things to it after you put initial volume)

  • Lag phase 

    • Interval between inoculation of culture and start of growth 

    • Time needed for biosynthesis of new enzymes to produce required metabolites before growth can start. 

  • Exponential Phase

    • Cells are healthiest here

  • Stationary Phase 

    • Death = Growth 

    • Either essential nutrient is used up and waste products accumulate (they are acidic and it accumulates and becomes toxic to bacteria)

      • Secondary metabolites 

        • Starts in this phase

        • Produced antibiotics that help survival in stressful situations with less food. Kills others 

  • Death Phase

    • Slower than exponential 

    • If incubation continues after cells reach stationary phase

4. Continuous Culture

• Continuous Culture 

  • Open system microbial culture of fixed volume (you can add more culture after)

  • Chemostat (container)

    • Common type of continuous culture device 

  • Allows you to control dilution rate to set up 

    • Steady State 

      • Cell density and substrate concentration stay constant over time. 

  • Purposes:

    • Mostly keeps them in exponential (or log) phase bc this is when they are happiest and can produce the most plasmids to for pharmaceuticals depending on bacteria but it could also be stationary phase

    • Whatever phase makes the product you want 

  • Experimental Purposes:

    • Study evolution and ecology etc. mutations will occur 

5. Growth Media and Laboratory Culture

• Different types of growth media with different nutrients for different bacteria. 

  • Defined Media

    • Chemical comp is known

  • Complex Media

    • Composed of digest of microbial, animal, or plant products (yeast or meat extract) 

    • Subclasses

    • Enriched media 

      • Complex media + highly nutritious materials

    • Selective Media 

      • Has compounds that selectively inhibit growth of certain microbes 

    • Differential Media

      • Has indicator (dye) to detect particular metabolic reactions during growth 

• Autoclave 

  • Sterilized by this

6. Measuring Microbial Growth

• 30-300 colonies is good for statistical analysis more or below 

• Microscopic Cell Count

  • Limitations:

    • Can’t distinguish living or dead without special stains 

      • Ex. DAPI (Blue) reacts with DNA 

    • Precision is difficult

    • Counts dead cells 

• Viable Counts

  • Report CFUs or Colony Forming Units instead of single cells 

  • Counts living cells

    • Pros: 

      • Quick and easy 

• Turbidimetric Measure of Cell Numbers

  • Spectrophotometer to measure cloudiness and molecules absorbing light. More cloudy + more bacteria 

  • Measures Optical Density (OD)

  • Pros:

    • Quick and Easy 

    • Typically doesn't kill culture 

    • Same samples can be checked repeatedly

  • Cons:

    • Microbes might form biofilms or clumps 

• Spectometometre 

  • Measures optical density (OD)

    • OD is proportional to cell number within limits 

1.B

2.B

3.D!!!!!!!

4.E!!!!!!!!

5.D

7. Controlling Microbial Growth

Decontamination 

• Treatment of something to be safe to handle 

Disinfection 

• Directly targets removing pathogens but not necessarily all microbes 

Heat Sterilization 

• Decimal Reduction Time (D) 

  • Amount of time required to reduce viability ten-fold

• Moist heat works better than dry heat (baking), you have to go to higher temps to equate the same killing as moist heat 

• Thermal Death Time 

  • Time to kill all death time

• Autoclave 

  • Sealed device that uses steam under pressure

  • Temp kills bacteria not high temperature 

Pasteurization

• Using precise controlled short burst of heat to reduce microbial load in heat sensitive liquids 

• Does not kill all organisms so different from sterilization 

Ultra Violet (UV)

• Radiation breaks DNA 

• Useful for decontamination of surfaces

• Ionizing Radiation 

  • Amount of energy to reduce viability 10-fold (D10) is analogous to D value

Radiation 

• Used for sterilization (where nothing is left alive) 

  • Cathode Ray Tubes

  • X-Rays

  • Radioactive nuclides

Filtration

• Avoids use of heat and sensitive liquids and gases 

• Used for heat sensitive liquids 

• Membrane filters 

7. Chemical Control of  Microbial Growth

Antimicrobial Agents 

• Are chemicals that kill or inhibit growth

  • -cidal = kills microorganisms 

  • -static = inhibits growth 

Minimum Inhibitory Concentration (MIC) 

• Smallest amount of an agent needed to inhibit growth of microorganisms

Sterilants, Disinfectants, Sanitizers, and antiseptics 

• Used to prevent growth on inanimate surfaces and external body surfaces 

  • Sterilants 

    • Destroy all microorganisms including endospores 

  • Disinfectants 

    • Used on surfaces to kill microorganisms but not necessarily endospores 

  • Sanitizers 

    • Reduce microbial numbers but not sterilize

  • Antispectic 

    • (Germicides)

    • Kill or inhibit microbial growth but are non toxic enough to apply to living tissue

    • I MISSED THIS DAY 

Obj: To understand how microorganisms cause disease by attaching to host tissues, colonizing and invading the body, and using virulence factors such as enzymes and toxins to damage host cells and evade the immune system

1. What Is Infection and Pathogenesis?

Microbial Edherence

• Infection

  • Microorganism is establishing and growing in host even if host is harmed or not

• Pathogen

  • Organisms that cause disease or tissue damage to host

• Pathogenicity

  • Ability of an organism to cause disease

2. Microbial Adherence (Attachment)

Structures used for attachment

• Capsules 

  • Protect bacteria from ingestion of white blood cells (leukocytes)

  • Capsule is sticky and has receptors to facilitate attachment to host tissue

• Fimbriae, Pili, Flagella

  • Surface proteins that help attachment

3. Colonization and Invasion

• Colonization

  • Growth of microorganism after tissue attachment

  • Typically starts with mucous membranes or tightly packed epithelial cells coated in mucus

  • Process begins at birth!

• Example of colonization

  • Tooth Decay

    • Streptococcus sobrinus and Streptococcus mutans, after initial contact, reproduce and form biofilm (plaque)

• Invasiveness 

  • Ability of pathogen to grow in host tissue at densities that inhibit host function 

    • Bacteremia

      • Presence of bacteria in bloodstream

    • Septicemia

      • Bloodborne systemic infection

    • Infection

      • Microorganism is established and grows in host 

4. Virulence

• Virulence

  • Ability of pathogen to cause disease

  • Measured using Lethal Does 

  • LD₅₀ (Lethal Dose 50%)

    • Amount needed to kill 50% of test subjects.

    • Lower LD₅₀ = more virulent.

• Attenuation

  • Decrease or loss of virulence 

  • Attenuated strains of various pathogens are valuable in clinic medicine because they are often used to produce viral vaccines 

5. Genetics of Virulence

Some bacteria have special genetic regions

• Pathogenicity islands

  • Clusters of virulence genes

  • Salmonella (SPI-1 and SPI-2)

• Plasmids

  • Extra DNA that contain upgrades, antibiotic resistance (R Plasmids)

6. The Compromised Host

• Compromised Host 

  • Susceptible to infection

• Opportunistic Infections

  • Caused by  organisms that do not cause disease in healthy hosts

• Nosocomial INfections

  • Infections acquired at hospital

7. Enzymes as Virulence Factors

Invasiveness requires pathogen to break down host tissue

• Hyaluronidase

  • Breaks down host tissue

• Coagulase and Streptoinase 

  • Manipulate clotting

  • Coagulase forms them

    • They form a blood clot before immune system can get to them

  • Streptokinase breaks them down

    • Breaks clots while immune system are away to keep growing 

8. Exotoxins

• Exotoxins 

  • Proteins secreted by bacteria 

  • Highly toxic 

  • Inhibit cell host function or kill host cells

• Three Main Types (They only work on cells with the right receptor)

  • AB Toxins(Two Parts)

    • Active(A) Domain

      • Does damage

    • Binding (B) Domain

      • Attaches to host cell

    • Examples

      • Diptheria Exotoxin

        • Blocks proteins synthesis

          • Can cause thick covering of back of throat leading to difficulty breathing, heart failure, paralysis

      • Botulinum Exotoxin

        • Blocks nerve signals

        • Causes paralysis

        • Effects diaphragm so you cant breath

      • Tetanus Exotoxin

        • Blocks nerve signalsCauses muscle spasms

        • Lockjaw

        • Can cause suffocation

      • Cholera Toxin (enterotoxin)

        • Effects small intestine

        • Massive secretion of fluid into intestinal lumen resulting in vomiting and diarrhea

  • Cytolytic Toxins

    • Destroy cell membranes causing cell lysis and death

    • Hemolysins destroy red blood cells 

    • Ex. staphylococcal toxin kills nucleated cells 

  • Superantigen Toxins

    • Overstimulate immune system because antigens trigger your immune system

      • Usually of T cells leading to cytokine storm 

      • Can lead to shock and death

      • Generally due to localized infection 

      • Mainly gram +ve bacteria 

        • Ex. Toxic Shock Syndrome 

          • Antigen has to be presented to Tcell which will kick off immune system and only only the t cell group will activate bc it recognizes the antigen. 

          • If the wrong antigen is presented to Tcell, then nothing happens

          • BUT if the wrong antigen is presented but the toxin superantigen is there it will fool the T cell and activate it. A lot ofT cells releasing cytokines that should not have been released will be

9. Endotoxins

• Endotoxin = LPS (Lipopolysaccharide)

  • Found in Gram - bacteria

  • Lipid A = toxic portion

    • Released when bacteria dies 

    • Causes fever

    • Septic Shock

    • Inflammation

  • Detected by 

    • Limulus amoebocyte lysate (LAL) test (horseshoe crab blood).

    • Test for LPS detection1.B

2.B

3.A

4.C

5.C