chapter 5 mab

What is the approximate number of bacteria found in or on the human body?

100 trillion

Which of the following is a characteristic of prokaryotic cells?

DNA is round and lacks histones

What is the approximate number of bacteria found in or on the human body?

100 trillion

Which of the following is a characteristic of prokaryotic cells?

DNA is round and lacks histones

What is the primary purpose of Gram staining?

Differentiate between Gram-positive and Gram-negative bacteria

What color does Gram-negative bacteria appear after Gram staining?

Pink

Which type of bacterial shape is rod-shaped?

Bacilli

What environment does a halophile thrive in?

Salty

Which microorganism was the first eukaryote to have its genome sequenced?

Saccharomyces cerevisiae

What is the function of pilli in bacteria?

DNA transfer

Which enzyme is produced by Vibrio bacteria?

Luciferase

Which of the following microbes is used to degrade resins and pollutants?

Pseudomonas stutzeri

What is bioprospecting?

Sampling the environment for useful microbes

Which gene produces bioluminescence in Vibrio bacteria?

Lux gene

What are extremophiles?

Microbes that survive in extreme conditions

What feature differentiates Gram-positive from Gram-negative bacteria?

Thickness of the peptidoglycan cell wall

What is the main component of the bacterial cell wall?

Peptidoglycan

Which yeast species is commonly used in batch processes for producing large quantities of products?

Pichia pastoris

Which of the following is NOT a microbial application?

Constructing buildings

What type of chromosomes are found in bacteria?

Circular and without histones

Which type of staining helps differentiate bacterial types based on cell wall structure?

Gram staining

How do Gram-positive bacteria appear under a microscope after Gram staining?

Purple

What is the purpose of the tag protein in cloning and expression techniques?

To indicate successful protein production

What bacterial feature is responsible for transferring DNA between cells?

Pilli

Which of the following is classified as a thermophile?

T. aquaticus

Microbial Enzymes

Used for food production, clothing, and research

Ex. - Taq DNA pol, Pfu DNA pol, Cellulase, Subtilisin(Bacillus subtilis), Amylase

Extremophiles

Some microbes are capable of surviving in extreme conditions

Able to survive extreme -

Salty environments

Halophiles

Heat

thermophiles

Acid/Basic

acidophiles/alkaliphiles

examples

T. aquaticus(bacteria), Methanopyrus kandleri(archaea)

Bioprospecting

Process of going out into the environment to sample for bacteria that may be useful for us

How do we get proteins of interest?

1. Gene for tag protein is attached to the gene for protein of interest(insert this into an expression vector

2. This rDNA creates a fusion protein

Tag protein

indicator that your protein is being produced in the cell

fusion protein

The protein product of a gene created by the fusion of two distinct genes or portions of genes.

Cloning and Expression Techniques

Lyse Cells

Homogenize cells to create extract

Put extract through column -

Put proteases through column to cut off protein of interest from tag protein

Affinity chromatography

column can have plastic beads that bind to "tag" protein

Expression vectors

1. Prokaryotic promoter

2. Gene for "tag" protein

3. Cloned gene of interest

Commonly used Tag proteins: luciferase, GFP, B-galactosidase and maltose binding protein

Bacteria - E. coli and B. subtilis

Reporter genes

Vibrio bacteria produce light via lux genes

Lux genes produce luciferas

Medical application: Tb test(Mycobacterium tuberculosis)

Bioluminescence: Luciferase

Luciferan gets turned into oxyluciferin

Happens because of an enzyme called Luciferase

Luciferan turns into oxyluciferin and light

The lux genes creates luciferase

lux genes

genes in E. coli (luciferase) that emit light (from Vibrio or Photobacterium)

Amount of bacteria in/on humans

100,000,000,000,000

Amount of your own cells

35,000,000,000,000

Prokaryotic

bacteria or archaebacteria

Eukaryotic

protista, fungi, plants, or animals

Microorganisms

Microbes are too small to be seen with the naked eye, and must be viewed with a microscope

Microbes include: bacteria, fungi, algae, viruses, and protozoa

Bacteria are the most abundant

Bacteria has existed for over 3.5 billion years

Less than 1% have been cultured/identified

Application of Microbes

save paintings; Pseudomonas stutzeri degrades resins and pollutants

Eukaryotic Microbes

Plant and animal cells, fungi(yeast)

Prokaryotic Microbes

Bacteria and archaea

Prokaryotic

DNA structure is usually round without histones

May contain plasmids

Lack membrane bound organelles

Contain a cell wall rich in peptidoglycan

Some bacteria contain and outer capsule made of carbs

Classified using gram staining

Use pilli to transfer DNA

Typically divide every 20 min ideally

Chromosomes

2-4Mb (mega bases)

pilli

Hollow tubes used to move cells or exchange DNA between bacteria by conjunction.

Gram Staining

Helps us differentiate between gram positive and negative bacteria

1. Treat them with crystal violet(CV)

2. Then apply iodine. Iodine(I) binds with the CV. CV-I

3. Then add alcohol wash. Gram positive keep the CV-I, while gram negative cells will lose the CV

4. Then apply safranin(S) - "counter stain," allowing you to visualize gram negative cells.

Gram positive

appear purple

Gram negative

appear pink

Peptidoglycan

cell wall

Gram positive cell wall

thick peptidoglycan layer

gram negative cell wall

thin peptidoglycan layer

Cell wall in bacteria

The cell wall in both cells determines if the CV-I stays or leaves, because cell wall is thin in gram negative cells, it can escape the cell

When safranin is added, it stains gram negative cells pink but doesn't affect Gram positive cells since they are already stained

Bacterial Shapes

3 common types: Cocci(coccus), bacilli(bacillus), spiral(spirochete)

Bacilli

rod shaped

Cocci

round

spirilli

corkscrew

Kingdom Fungi: Yeast

Beer recipes from ancient babylonians(4300 B.C.) were discovered using yeast to ferment to create beer

Yeast are frequently used to create medicine - antibiotics & cholesterol lowering drugs

Can grow in anaerobic (without oxygen) or aerobic conditions (with oxygen)

S. cerevisiae

first eukaryote to have its genome sequenced

Pichia pastoris

used for batch processes(producing large amount) because of its many promoters

Yeast reproduction

budding or fission

Use of Microbes as Model Organisms

Bacteria:

Studying molecular biology

Genetics

Biochemistry

Biotechnology

Yeast:

Chromosome structure

Gene regulation

Cell division

Cell cycle control

Gram + vs. Gram

Innermost layer- Plasma membrane

Middle layer - Cell Wall

Outmost layer - Capsule

Gram +

Innermost plasma membrane, thick peptidoglycan cell wall, outer capsule.

Easily treatable with antibiotics since lacking 2nd cell membrane

Stains purple/violet after Gram Stain

Gram -

Innermost plasma membrane, thin peptidoglycan cell wall, another plasma membrane, outer capsule

Not easily treatable

Stains red/pink after Gram Stain

Gram Stain

Identify type of infectious bacteria

Treatments differ based upon outcome