Microbiology: Chapter 10

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Classification of Microorganisms

Phylogeny / Systematics

The study of the evolutionary history of organisms. Hierarchy of taxa reflects evolutionary / phylogenetic relationships.

Organism similarities

All organisms are composed of cells surrounded by a plasma membrane, use ATP for energy and store genetic information in DNA. Result of evolution or descent from common ancestor.

Taxonomy

The science of naming and classifying living organisms. Put organisms into categories / taxa to show degrees of similarities among organisms. Similarities due to relatedness through evolution. Also used to facilitate research, scholarship and communication.

Classification into three domains

Organisms are classified by cell type in the three domain systems.

1. Living organisms are classified into three domains (Bacteria; Archea and Eukarya) based on similarities in ribosomal RNA (rRNA). rRNA mutates less over time compared to DNA. (Also look at tRNA & mRNA but to lesser extent).

2. In addition to rRNA, the 3 domains differ in cell membrane lipid structure.

3. Domains also differ in antibiotic sensitivity / cell wall components

Endosymbiosis

Endo (inside); symbiosis (2 organisms living together - relationship can be good or bad)

Endosymbiotic theory: A model for the evolution of eukaryotes which states that organelles arose from prokaryotic cells living inside a host prokaryote.

1.4 billion years ago eukaryotic cells evolved from prokaryotes. (endosymbiotic bacteria/proK developed into organelles & gave rise to mitochondria and chloroplasts)

Why euKs developed from ProK?:
Mitochondria: have same size and shape as bacteria with circular DNA (same as bacteria). Table 10.2

Prokaryotic cell, mitochondria and chloroplasts divide / replicate through binary fission (circular genome divide). Have 70S ribosomes, whereas eukaryotes have 80s ribosomes throughout cytoplasm and on RER.

Domains: 1. Eukarya

Fungi, Protista, Animals & Plants

Domains: 2. Bacteria / Eubacteria

(Prokaryotes) Includes all of the pathogenic prokaryotes as well as many of the nonpathogenic prokaryotes found in soil and water.

Also include Cyanobacteria: Oxygen-producing photoautotrophic bacteria / prokaryotes that can photosynthesize. (Used to be called blue-green algae and classified into protista kingdom)

Measurement of cell wall difference:
Gram+: Thicker peptidoglycan cell wall
Gram-: Thinner peptidoglycan cell wall

Domains: 3. Archeae

(Prokaryotes) Around for only 20+ years. Some have no cell wall. Those with cell wall contains no peptidoglycan. In question, whether or not they cause disease. They live in extreme environments.

1. Methanogens: Anaerobes that produce methane (CH4) from CO2 and H

2. Halophiles: Require high concentration of salt for survival

3. Hyperthermophiles: Grow in extremely hot environments

4. Thermoacidophiles: Live in extreme high temperatures and acidic environment

Characteristics: 1. Eukaryotes

Cell Type: Eukaryotic

Linear DNA tightly coiled with histo proteins (histones). 80s Ribosomes. Grow via mitosis.

Cell Wall: Varies in composition; contains carbohydrates

No antibiotic sensitivity.

Characteristics: 2. Archeae

Cell Type: Prokaryotic. Circular DNA with no nucleus / nucleur membrane. DNA contains histones (like EuKs). 70s Ribosomes.

Cell wall: if cell wall, no peptidoglycan

Grow via binary fission

Characteristics: 3. Bacteria

Cell type: prokaryotic. Circular DNA (no histones), 70S Ribosomes.

Grow via binary fission.

Cell wall contains peptidoglycan.

Antibiotic sensitivity: Yes

Taxonomy of Eukaryotes

(Eukaryotic species: A group of closely related organisms that breed among themselves)

Kingdom
Phylum
Class
Order
Family
Genus (Consists of species that differ in certain ways but related by decent / genetically)
Species: Eukaryotic species is a group of closely related organisms that can interbreed.

Particular organism (or species) has a genus name & specific epithet, and belongs to a family, order, class and phylum. Phyla related to each other make up a kingdom, and related kingdoms are grouped into a domain.

Taxonomy of Prokaryotes

Domain: Bacteria & Archea
Phylum (no kingdom)
Class
Order
Family
Genus
Species: Prokaryotic species is a population of cells (asexual) with similar characteristics.
Sub-species

Culture

Bacteria growing in media (agar, jelly, broth) over particular time.

Clone

Pure culture. A population of cells derived from a single parent cell. All cells in a clone should be identical.

Sub species / strain / serotype / serovars / biovars

A variation within a species.

Genetically different cells within a clone. In some cases, pure cultures of the same species are not identical in all ways. Each such group is called a strain. Strains are identified by numbers, letters, or names that follow the specific epithet (eg. subspecies: E. Coli 0157:H7)

Viral Classification

Not classified as part of any of 3 domains (non-living)
Use anabolic machinery within living host cells to multiply. A viral genome can direct biosynthesis inside a host cell, and some viral genomes can become incorporated into the host genome.
Obligatory intracellular parasites.
No cell structure or organelles
Core with RNA or DNA
Capsid: protein coat

Much smaller than bacteria, only visible with electron microscope.

Microbe Classification: Morphology

Structure.
Colony morphology: How it appears in petridish.
Tells us little about phylogenetic relationships, but can be useful to identify differences in structure such as endospores or flagella.

Microbe Classification: Staining

(Differential staining) Most bacterie are either gram+ or gram-. Microscopic examination of gram stain is used to obtain information quickly in clinical environment. Other types of stainning done after gram stain.

Microbe Classification: Biochemistry

Testing to see if they use a chemical (eg. glucose) or ferment eg. lactose. If they do they have an enzyme (enzymatic activity) for it and therefor the required gene. (See figure 10.8)

Rapid identification methods: Enterotube performs several biochemical tests simultaneously, can identify bacteria within 4-24 hours. One tube containing media for 15 biochemical tests is inoculated with an unknown enteric baterium. (fig 10.9)

Microbe Classification: Serology

The branch of immunology that studies blood serum and antigen-antibody (Ab-Ab) reactions in vitro.

Microorgs are antigenic: stimulate animal's body to form antibodies.

Antiserum: blood-derived fluid containing antibodies. Unknown bacteria can be tested against known antisera for identification of bacteria.

Serological testing can differentiate between microbial species and serotypes (different strains of same species).

Types of serological tests include: ELISA & Western Blot

Microbe Classification: Serology - ELISA

ELISA (Enzyme-linked immunosorbent assay): Widely used because fast and read by computer scanner. figure 10.11

1. Direct ELISA: Known antibodies are placed in microplate, and unknown bacteria added. A reaction between known antibodies and bacteria provides identification of the bacteria. (look for antigen)

2. Indirect ELISA: looking for antibody. Antigen stays hidden, while the antibodies show up in the bloodstream (HIV, Lyme)

Microbe Classification: Serology - Western Blot

(figure 10.12) Also used to identify antibodies in patient's serum. (eg. HIV, Lyme)

1. Proteins from known bateria or virus are separated by an electric current in electrophoresis. (Proteins (- charged) move at different rates based on charge and size when the gel is exposed to the electric current.

2. Proteins transferred to nitrocellulose filter by blotting.

3. Patients serum washed over filter. If patient has antibodies to one of the proteins in the filter, the antibodies and protein will combine. Anti-human serum linked to an enzyme is then washed over filter (Ab-Ag complex visible as colored band on filter after adding enzyme's substrate)

4. Test is read. If tagged antibodies stick to filter, evidence of presence of particular microorg. has been found in patient's serum.

Microbe classification: Phage-typing

(figure 10.13)
Used to trace sources of infection by: determining which phages a bacterium is susceptible to. Sources of food-associated infections can be traced by phage typing.

Bacterial phages: bacterial viruses that cause lysis of the bacterial cells they infect. Viruses are specific to certain species or strains.

1. Begin with bacterial lawn (plate totally covered with bacteria growing on agar)
2. Drops of different phage types are placed on the bacteria.
3. Wherever phages are able to infect and lyse the bacterial cells, plaques appear (clearing areas in bacterial growth where virus killed bacteria)

Microbe classification: Fatty-acid profile

Many bacteria synthesize variety of fatty acids. FAs are constant for particular species. Used for classification.

Microbe classification: Flow-cytometry

Used to identify bacteria in a liquid sample (cyder, milk) without culturing the bacteria.

Fluid forced through small opening. Detects presence of bacteria by counting number of particles, their size, and detecting difference in electrical conductivity between cells and surrounding medium. Results analyzed by computer.

eg. Milk tested to detect Listeria by labeling listeria antibodies with fluorescent dye and added to milk. Flow cytometer records fluorescence of antibody labeled cells.

Microbe classification: DNA-fingerprinting

(figure 10.14)
Uses restriction enzymes to cut / chop DNA everywhere a specific base sequence occurs (always the same), produces restriction fragments.

1. DNA from different microorgs treated with same restriction enzyme, which produces restriction fragments. (DNA placed in wells)

2. Restriction fragments are separated by electrophoresis (apply electrical current to gel to separate the fragments by size and electrical charge)

3. Comparison of results. The more similar the paterns (DNA fingerprints), the more closely the organisms are related, or if different, no relation.

Microbe classification: PCR

(Polymerase Chain Reaction)
Used if microbe cannot be cultured by conventional methods. Used to increase amount of microbial DNA to levels that can be tested by gel electrophoresis.

Sample put through PCR, adding primers & DNA polymerase. Make copies of genetic material - DNA is amplified.

Microbe classification: rRNA sequencing

rRNA sequencing of bases of different organisms are analyzed and compared. rRNA is used because it mutates less than DNA.

Microbe classification: DNA hybridization (Nuclein Acid Hybridization)

(Figure 10.15)
Uses complimentary base pairing (single stranded DNA)

1. Heat is used to seperate DNA strands of 2 organisms

2. Single strands are cooled slowly - DNA single strands from 2 different organisms combine.

3. Cool to allow renaturaion of double-stranded DNA

4. Amount of pairing between DNA strands from different organisms analyzed (hybridization). Look at G+C ratio. If difference between G+C ratio is 10% or less then organisms are related. If difference is larger, organisms are unrelated.

Complete hybridization (combining of DNA strands) - Organisms are identical
Partial hybridization - organisms are related
No hybridization - organisms are unrelated

Microbe Classification: DNA hybridization - DNA probes

(Figure 10.16)
Type of DNA hybridization.
DNA fragments marked with fluorescent dye & separated into single strands (fluorescent probe). Added to unknown bacteria. Hybridization occurs and presence of fluoresence indicates presence of bacteria in question.

Microbe Classification: DNA hybridization - DNA chips

(Figure 10.17)
Several probes run at same time.
DNA chip containing many single stranded sequences. Unknown DNA from patient seperated into single strands and labeled with fluorescent dye. Unknown DNA inserted into chip. Tagged DNA will bind only to complementary DNA on chip. Hybridization between the probe DNA and DNA in the sample is detected by fluoresence. Analyzed by computer.

Microbe Classification: DNA hybridization - Southern Blot

(Figure 9.16)
Use nitrocellulose blotter and gel electrophoresis.

1. Electrophoresis seperates DNA fragments according to size into bands. Bands made visible with staining.

2. DNA bands transfered to nitrocellulose filter by blotting.

3. Filter then exposed to radioactively labeled probe for specific gene. Probe hybridize with sequence present on gene.

4. Filter exposed to x-ray film and fragment with gene (band) is seen on developed film.

Microbe Classification: Dichotomous Keys (Putting classification methods together)

(figure 10.8)
(dichotomous - cut in two)
Used for identification and not for phylogenetic / evolutionary relationships. Many experiments are run to narrow down the type of organism.

Microbe Classification: Cladograms
(Putting classification methods together)

(Figure 10.19)
(Clado - branch)
Uses rRNA sequencing to create maps that show evolutionary relationships among organisms. Similarities are compared (all with one common ancestor)

Exam Question: Look at clydogram and determine which orgs have most in common genetically.

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