Microbiology Test 2 Review PDF

Preview

Summary

Download Microbiology Test 2 Review PDF

Description

Early bacterial genetic researchers focused more on the microbes with practical importance (ex. Pathogenic bacteria). Example: E coli in our gut is useful, OR salmonella which makes us sick Researchers today are more focused on understanding the genetic potential of microbes. Bacteria are used for making drugs (ex. Streptomyces, a genus of bacteria, gives us antibiotics, Pseudomemes, is pathogenic and is used in cleaning industrial contaminants.)

-

-

Growth → multiply/increase in number of cells NOT when they grow in size. * Bacteria are ideal candidates for genetic research because… - They have 1 chromosome/copy of gene → easy detection of mutants - If a mutation occurs → show a phenotype b/c 1 chromosome. Different strains of bacteria are clones of a certain species. Early studies, prototrophs and nutritional mutants were used. -

- grow on normal medium NO MUTATION need to add additional components to grow mutant strain Allowed the study of one gene based on its inability to produce a particular nutrient Leterbergs experiment (Rate of reversion)

Bacteria grow into isolated colonies on plates, you can reproduce these colonies by “Stamping” the original plate with a cloth and then stamping empty plates with the same bloth . Bacteria from each colony are picked up on the cloth and then deposited on the new plates by the cloth. Hypothesis : Antibiotic resistant strains of bacteria surviving an application of antibiotics had the resistance before their exposure to the antibiotics, not as a result of the exposure. Experiment : Original Plate - Bacteria are spread out on a plate, called the “original plate.” Original Plate- They are allowed to grow into several different colonies Stamped plate with penicillin- The layout of colonies is stamped from the original plate onto a new plate that contains antibiotic penicillin Stamped plate with penicillin- Colonies X and Y on the stamped plate survive. They must carry a mutation for penicillin resistance Original plate with penicillin- The leaderboards set out to answer the question; did the colonies on the new plate evolve antibiotic resistance because they were exposed to penicillin? NO When the original plate is washed with penicillin the same colonies X and Y live - even though they have never encountered penicillin before

10(how many colonies are growing)/10^8 = 10^-7 100(how many

colonies are growing)/10^8=10^-6 Reversion Rate= 10^-7- 10^-6 ****** GO TO OFFICE HOURS TO UNDERSTAND THIS

Organization of bacterial genomes → the genetic material | Most is transcribed → The information of a strand of DNA is copied into a new molecule of mRNA - Single circular chromosome and plasmids (if any) - Plasmid → A circular DNA molecule that replicates independently from chromosomal DNA, these genes are used for survival - may be used for symbiosis - causes lyme disease,  & have a linear chromosomes - not circular When the bacterial plasmid/chromosome can replicate from a single origin of replication may be present → a type of virus that attacks only bacteria

-

-

The inability of two plasmids to coexist stability over a number of generations in the same bacterial cell line. Closely related plasmids tend to be incompatible but distantly related plasmids tend to be compatible - Replication will treat two plasmids as one if they have the similar origin of replication → one will keep replicating while the other will not be able to replicate and will stay as one plasmid. One plasmid “loses out”, not being replicated

Mutations may cause loss of function of a gene, regain function of gene that was previously mutated, and change in the phenotype. can create a mutation in bacteria

Changes in genes are often visible by changes in the  - If it contains capsules you can mutate the capsule and see that it looks different. Microbial geneticists work to compare wild-type strains and mutant strains of bacteria with the goal to - The genotype of an organism describes its collection of alleles of a given set of genes “Other” → Two or more alternative forms of a gene that arise from a mutation General designation “rules” - A is given a  , followed by a  to separate genes in the same pathway (ex. lacY vs lacZ ) - A is given the same  but with the and  (ex. LacZ) Detection of Mutants (CFU, colony forming units - potential to form colonies) -

Use of a growth medium that will inhibit microbes lacking the desired genes Antibiotic selection is commonly used for phenotypic mutation in bacteria

Duplicate plates are created - The first is under full nutritional support - A colony grows on this fully supported plate - The second lacks a particular nutrient - Colony does not grow on this partially supported plate → a mutation occurred Duplicate plates are created by the process of Evolution in a test tube - Not all mutations are bad or slow! Lenskirs work in 1988 illustrated the evolution of  in a period of 75 days, increasing in fitness - used carbon tube as glucose - Cultures were allowed to grow, given fresh nutrients and space (without selective pressure) - Cultures of this extended generational time were compared to stored original cultures that did NOT have these conditions The ability to grow in culture was enhanced over time This is evidence of - the ratio of red to white colonies, the survival or reproductive rate of a genotype or phenotype relative to the maximum survival or reproductive rate of the other genotypes in the population - The molecular changes that cells undergo in response to environmental stresses How is DNA cloned?

-

Can be used to make recombinant DNA Allow cutting of specific DNA pieces (a required prerequisite to moving or copying DNA fragments)

-

Each restriction enzyme recognizes a restriction site and cuts it, often the cuts are asymmetrical Similar ends of cut DNA can be paired together Paired ends can be tied, or ligated, by 

Restriction Enzyme

Source

Restriction Site

Cleavage Result

EcoRI

Escherichia coli

5’ - GAATTC - 3’ 3’- CTTAAG - 5’

5’ - G ATC - 3’ 3’ - CTTAA G-5’

BamHI

Bacillus amyloliquefaciens

5’-GGATCC - 3’ Palindrome- Same sequence on other strand but backwards 3’ - CCTAGG - 5’

5’- G GGATCC-3’ 3’-CCTAG G-5’

HindIII

Haemophilus influenzae

5’- AAGCTT -3’ 3’-TTCGAA-5’

5’ - A AGCTT-3’ 3’-TCGA A-5’

SmaI

Serratia marcescens

5’-CCCGGG-3’ 3’-GGGCCC-5’

5’-CCC 3’-GGG

-

-

-

GGG-3’ CCC-5’

can be used to “clone” or make many copies of a bacterial gene of interest Vectors are used to insert a recombinant DNA molecule into a recipient host bacterial cell

Normal medium usually used - LB Cut fragments from two plasmids carrying antibiotic resistance genes with the same restriction enzyme, followed by ligation with DNA ligase - (stitch back pieces of DNA) Repairs single and double strand breaks that occur during DNA replication After inserting the recombinant plasmid into bacteria, the strain exhibited traits from both plasmids. Today, there are a number of “common” plasmid vectors with desirable traits for easier gene cloning:

Can we make screening for desired insertions easier ? - X-gal system = visual blue / white colony growth - Blue/white selection- dont need any drug - Bacteria continuously making LacZ - LacZ makes LacZ alpha as long as its intact make beta galactosidase- give blue color to bacterial colonies= presence of x gal - No lacZ alpha - no beta galactosidase - White is gene of interest - Phage (cloning) vectors mix viral DNA with the fragment of interest - Lysogenic lambda phage can carry ~20 kb fragments - Lambda phage effects E coli - Phages vs plasmids - plasmid is limited can carry up to 15kb at most, phages can infect themselves by just mixing, big pieces can be used by this -

Cosmids are phage genomes that delete nearly all of the phage DNA, leaving more room for fragments - Only critical phage c os  packaging recognition sites are left - Other elements include multiple cloning sites and an antibiotic selection marker - Cosmids can typically carry 35-45 kb fragments (of gene of interest)

Vector- 10 kb Plasmid - 15 kb max Phages- 24 kb Cosmids- 45 Kb

-

Methods for introducing DNA into microbes often results in disruption of genes → allows for the study of effects of their loss on phenotypes Genome- complete set of DNA, including all of its genes, plasmids(bacteria), genes including mitochondria(humans) Genetics- The study of individual genes and their function - DNA sequencing- the process of determining the nucleic acid sequence Genomics- Deals with collective properties and quantification of different genes- recombinant DNA

Genome sequencing

Genomic analysis of gene expression Genomics has been spurred by the development of recombinant DNA protocols - However, this has created several new needs for, - Improved DNA sequencing techniques - Formats for storage of very large data sets - Tools for analysis of large data sets generated -

Walter Gilbert developed a chemical degradation method

-

Sanger, or dideoxy sequencing method DNA sequencing requires three steps:

-

DNA polymerases require a

Complementary strands are held together by  Nucleotides present on the same strand are linked by  In DNA and RNA, the phosphodiester bond is the linkage between the 3 inch carbon atom of one sugar molecule and the 5 inch carbon atom of another, deoxyribose in DNA and ribose in RNA The phosphodiester linkage between two ribonucleotides can be broken by alkaline hydrolysis, whereas the linkage between the two deoxyribonucleotides is more stable under these conditions - By placing dideoxynucleotides (lacking that free 3’ OH group) Into the DNA synthesis fixture, the process is terminated with the distinct labeled end point nucleotide - Gel electrophoresis can separate the fragments of the different length and detect which labeled nucleotideIs on the end of each fragment, providing a sequence - Break DNA by breaking the phosphodiester linkage - RNA is unstable due to the 2 prime hydroxyl group which is absent in DNA - If the 3 prime hydroxyl group is removed = deoxyribose The sanger method of DNA sequencing

-

Automated methods using fluorescent  labels instead of radioactive labels are safer, cheaper, and easier - Sequences of 700 to 1000 bases obtained cheaply in hours - Longer sequences obtained by “primer walking”, using repeated rounds of sequencing with primers complementary to the end of the last segment sequenced Sanger method of DNA sequencing - Most common approach for DNA sequencing - Invented by Frederick Sanger - 1977 - Nobel prize - 1980 - Also termed as the chain termination or dideoxy method

Whole-genome  - Attempts to sequence entire genome in one setup - DNA fragments are sheared and sequenced - Software aligns sequences - May need 10x total genome length to do so successfully Shotgun sequencing was based on the sanger sequencing method : this was the most advanced technique for the sequencing genomes from about 1995 - 2005. The shotgun strategy is still applied today, but now we use other sequencing technologies, called next-generation sequencing.

-

Detects addition of a nucleotide at the end of a synthesized strand of DNA by production of light One by one, 2ppi is produced APS - adenosine phosphosulfate = ATP = luciferase = light Faster and cheaper than the Sanger method

These technologies produce shorter reads (anywhere from 25-500 bp) but many hundreds of thousands or millions of reads in a relatively short time (on the order of a day). This results in high coverage, but the assembly process is much more computationally intensive. These technologies are vastly superior to Sanger sequencing due to the high volume of data and the relatively short time it takes to sequence a whole genome. - Requires a lot of computing, need to put sequences in order

-

Illumina (solexa) sequencing* Nanopore DNA sequencing* Single molecule real time (SMRT) sequencing*

-

DNA nanoball sequencing* SOLiD sequencing*

-

Works by reading the nucleotide sequences at the single molecule level Very precise, extremely fast Don't need to know

-

Analysis of large data sets of sequencing data - Annotation of genomes helps researchers identify open reading frames (ORFs) - ORFs allow us to better determine the start and stop points for a given gene(protein) - Sequencing speed and cost is incredible today - but it only gives us raw information - Functions of genes need to be determined , not just sequences - this is the goal of 

How is gene expression measured using genomics tools? -

A genomic library is a collection of cloned DNA fragments that  represents the entire genome of an organism - Bigger size of genome- more fragments needed The number of cloned fragments needed to encompass an entire genome can be determined by the formula N= ln (1-P) / ln (1-f) ******remember equation, no math equation N= number of clones required P= desired probability of generating a complete library F = the average size of the each clone divided by the total size of the genome Two bacteria category- pathogenic and nonpathogenic Processes used by pathogens to produce disease ***remember Human requirements - food and shelter, same with microorganisms Best food and shelter is the human body Commensal bacteria= good bacteria Pathogenic bacteria= bad bacteria because it harms the body

What are common features of bacterial pathogens? - *virulence factors = pathogen products that enhance their ability to cause disease

-

Act in a common way, all like to attach first, overcome host defense and obtain nutrients by damaging tissues and replicating

Some major virulence factors of selected pathogens Organism:

Disease:

Virulence Factor:

Action:

**Bordetella pertussis REMEMBER

Whooping cough

Fimbriae Pertussis toxin Invasive adenylate cyclase

Attachment Disrupts cell ion balance Disrupts cell ion balance

****Escherichia coli O157:H7 REMEMBER

Hemorrhagic colitis and kidney failure

Intimin Tir Type III secretion system Shiga toxins*

Attachment Receptor for attachment Injects Tir for attachment Stops translation in host cells

****Helicobacter pylori H pylori REMEMBER

Gastritis, ulcers

Urease Vacuolating cytotoxin A (VacA) Flagella* Cytotoxin-associated antigen (CagA)* CagA type V secretion system

Neutralizes gastric acid Host cell death, inflammation Transport through mucus Disrupts host cell cytoskeleton Injects CagA

*****Neisseria gonorrhoeae REMEMBER

Gonorrhea 80% females are asymptomatic Men with this will have high chance for prostate cancer

Fimbriae IgA protease LOS lipooligosaccharide(a form of endotoxin)

Attachment and immune evasion Destruction of IgA antibody Evokes inflammatory damage

****Streptococcus pneumoniae REMEMBER

Pneumonia, meningitis

Capsule Pneumolysin Autolysin

Anti-phagocytic Forms pores in host cells Lysis of bacterial cell to release peptidoglycan(produces inflammation)

*****Streptococcus pyogenes REMEMBER

Various skin, throat and systemic infections

Capsule M protein Hyaluronidase Streptokinase

Anti-phagocytic Prevents binding by antibody Degrades connective tissue Degrades fibrin clots

-

First virulence factor = attachment Specialized pili with a sticky tip - The tip binds to a specific receptor on the host cell - Acts as a “probe” to get beyond the repulsing negative charge on the host cell - Can be altered by some microbes to evade immunity

-

-

Endotoxins are a part of the cell wall structure and induce inflammatory responses - Lipopolysaccharide (LPS) in gram negative cells - Lipoteichoic acid (LTA) in gram positive cells Exotoxins are released outside the producing cells - A-B toxins : B subunit binds to host cell receptor. A subunit has a negative action inside the cell - Cytotoxins : Toxins that directly act on host cells - Cytolysins : Act on plasma membrane - Superantigens: Nonspecifically stimulates T cells to secrete large amounts of cytokines which can damage host cells -

Lipopolysaccharide (LPS) is the most common endotoxin** It has three parts : - O-antigen (often strain-specific, can be used for serotyping)** - Core polysaccharide *** - Lipid A (Causes inflammatory response)***

Repeating units of polysaccharide Strain specific Target of immune response Used for serotyping Various sugars with side chains Genus or species- specific Core sugars, inner and outer core Innermost component of LPS - heat stable LPS Hydrophobic nature allows it to anchor the LPS to the outer membrane Lipid component of endotoxins are responsible for gram - bacteria toxicity Unusual fatty acids Ex. Mycelia meningitis No O-antigen= LOS instead of LPS

Endotoxin and Lipoteichoic Acids - Similar in nature to LPS but found on gram-positive cells - Capable of inducing inflammation Biological effects of endotoxins and lipoteichoic acids Effect

Protective activity

Fever

Inhibition of pathogen replication, Increases in immune cell activities

Complement Activation

Lysis by MAC formation, induction of inflammation

Inflammation

Transport of immune cells and molecules to site of infection

B-cell proliferation

Antibody production

IFN - y expression from T cells

Activation of macrophages and NK cells

Stimulation of clotting cascade

Prevention of pathogen spread

In small quantities it's actually useful -

Bacterial toxins: A-B toxins*** remember chart - B portion binds to a receptor on a host cell - A portion has enzymatic activity inside host cells - Many pathogens secrete A-B toxins - Corynebacterium diphtheria A-B toxin - A subunit inactivates EF2, abolishes translation, prevents protein synthesis - Shiga A-B toxin- affects kidneys in humans, lack in cattle, sheep and deer. - Produced by strain of E coli O157:H7(very toxic strain) represents capsule and flagella

-

Botulism toxin - Prevents muscle contraction very very toxic - Tetanus toxin - Produces continuous muscle contraction SNARE(snap receptors) proteins in host cells are used to release neurotransmitters*** Three syntaxin, snaptoxin and snap25 Some A-B neurotoxins cleave the SNARE proteins, preventing neurotransmitter release

- Work on plasma membranes of cells - “Membrane damaging toxins” (MDTs) describes the essential actions of cytolysins - More than ⅓ of all -

bacterial protein toxins Genetic structures of 70 cytolysins has been studied Hemolysins are a classic example, lysing(breaking down) red blood cells= hemolysis - Lysis pattern can be used as an identifier

-

Three Types: - **Alpha hemolysis : when the hemolysins partially break red blood cells (Green color) - Strep Pneumonia - **Beta: completely break down (red color) - Strep Pyogenes - **Gamma: No hemolysis (still growing) - EF, still very toxic -

-

-

-

Attack eukaryotic cells’ bilayer membranes by dissolving their phospholipids. C. perfringens x toxins, S. aureu...