micro lecture ch10

taxonomy-the science of classification of organisms, with the goal of showing relationships among organisms; also provides a means of identifying organisms

taxon-(plural taxa) subdivisions used to classify organisms, e.g., domain, kingdom, phylum

phylogeny-(systematics) the study of the evolutionary history of organisms

• The hierarchy of taxa reflects evolutionary, or phylogenetic, relationships
• 

The three domains differ in membrane lipid structure, transfer RNA molecules, sensitivity to antibiotics, and differences in rRNA.

• bacteria domains-bacteria with peptidoglycan; unicellular microorganisms found in every habitat on Earth; many bacteria are helpful just as some can be harmful; remember that the Gram Negative bacteria are more related to each other and Gram Positives are the same way
• cell type: prokaryotic
• cell wall: contains peptidoglycan
• membrane lipids: composed of straight carbon chains attached to glycerol by ester linkage
• First amino acid in protein synthesis: Formylmethionine
• Antibiotic sensitivity: yes
• rRNA loop- present
• common arm of tRNA- present

• archaea domains-bacteria with unusual cell walls and often live in extreme environments
• Not known to cause disease in humans or animals
• Includes: Methanogens (largest group of Archaea; produces methane as a metabolic byproduct; lives in guts of animals that use cellulose as their main foods), Extreme halophiles (extremely salt loving), Extreme thermophiles (extreme heat loving)
• cell type: prokaryotic
• cell wall: varies in composition; contains no peptidoglycan
• membrane lipids: composed of branched carbon chains attached to glycerol by ester linkage
• First amino acid in protein synthesis: Methionine
• Antibiotic sensitivity: no
• rRNA loop- lacking
• common arm of tRNA- lacking

• eukarya domains-plants, animals, fungi and protists
• cell type: eukaryotic
• cell wall: varies in composition; contains carbohydrates
• membrane lipids: composed of straight carbon chains attached to glycerol by ester linkage
• First amino acid in protein synthesis: Methionine
• Antibiotic sensitivity: no
• rRNA loop- lacking
• common arm of tRNA- present

• rRNA loop binds to ribosomal protein; found in all bacteria
• Common Arm of tRNA is a sequence of bases in tRNA found in all eukaryotes and bacteria: guanine-thymine-pseudouridine-cytosine-guanine

• Closely related strains, groups of bacteria derived from a single cell, constitute a bacterial species
• -a population of cells with similar characteristics
• -clone: population of cells derived from a single cell
• -strain: genetically different cells within a clone

• Eukaryotic organisms may be classified into the Kindom Fungi, Plantae, or Animalia
• Eukaryotic species are a group of closely related organisms that breed among themselves

Viruses are not classified as party of any of the three domains. Viruses are not composed of cells, and they use the anabolic machinery within living host cells to multiply. A viral species is a population of viruses with similar characteristics that occupies a particular ecological niche (habitate or home; who they infect).

In a world inhabited by millions of living organisms, biologists must be sure they know exactly which organism is being discussed. We cannot use common names bc the same name is often used for many different organisms in different locales. Plus, local languages are used for common names. Because common names can be misleading and are in different languages, a system of scientific names, referred to as scientific nomenclature, was developed in the eighteenth century

culture-bacteria grown at a given time in media; used to understand their metabolism and ecological role

clone-a population of cells derived from a single parent cell. All cells in the clone should be identical

strain-a group of bacteria derived from a single cell is called a strain. Not all cells in here are identical.

The Kingdom Fungi includes unicellular yeasts, multicellular molds, and macroscopic species such as mushrooms. To obtain raw materials for vital functions, a fungus absorbs dissolved organic matter thru its plasma membrane. The cells of a multicellular fungus are commonly joined to form thin tubes called hyphae. The hyphae are usually divided into multinucleated units by cross-walls that have holes, so that cytoplasm can flow between the cell-like units. Fungi develop from spores or from fragments of hyphae

The Kingdom Plantae (plants) includes some algae and all mosses, ferns, conifers, and flowering plants. All members of this kingdom are multicellular. To obtain energy, a plant uses photosynthesis, the process that converts carbon dioxide and water into organic molecules used by the cell

The kingdom of multicellular organisms called Animalia (animals) includes sponges, various worms, insects, and animals with backbones (vertebrates). Animals obtain nutrients and energy by ingesting organic matter thru a mouth of some kind

mostly unicellular organisms; these organisms are currently being assigned to kingdoms

A classification scheme provides a list of characteristics and a means for comparison to aid in the identification of an organism. Once an organism is identified, it can be placed into a previously devised classification scheme.

• Microorganisms are identified for practical purposes-for example, to determine an appropriate treatment for an infection.
• They are not necessarily identified by the same techniques by which they are classified.

• Some identification methods are:
• Morphological characteristics: useful for identifying eukaryotes
• Differential staining: Gram staining, acid-fast staining; shows morphological characters
• Biochemical tests: determines presence of bacterial enzymes; based on food sources or biproduces
• Serology-antigen response
• DNA base composition- DNA fingerprint comparisons

• Dichotomous Key: (two choice key)
• 

• Bergey's Manual of Determinative Bacteriology has been a widely used reference since the first edition was published in 1923.
• This manual does not classify bacteria according to evolutionary relatedness but instead provides identification (determinative) schemes based on such criteria as cell wall composition, morphology, differential staining, oxygen requirements and biochemical testing.

The standard reference for lab identification of bacteria

Morphological (structural) characteristics have helped taxonomists classify organisms for 200 years. Cell morphology tells us little about phylogentic relationships. However, morphological characteristics are still useful in identifying bactiera. For example, differences in such structures as endospores or flagella can be helpful

• differential staining can tell us if the bacteria is gram neg or gram pos.
• Microscopic examination of a Gram stain or an acid-fast stain is used to obtain info quickly in the clinical environment

Enzymatic activities are widely used to differentiate bacteria. Even closely related bacteria can usually be separated into distinct species by subjecting them to biochemical tests, such as one to determine their ability to ferment an assortment of selected carbohydrates.

• Serological testing can differentiate not only among microbial species, but also among strains within species.
• Strains with different antigens are called serotypes, serovars, or biovars.

Serological tests, involving the reactions of microorganisms with specific antibodies, are useful in determining the identity of strains and species, as well as relationships among organisms. ELISA and Western blotting are examples of serological tests.

• Phage typing is a test for determining which phages a bacterium is susceptible to.
• Bacteriophages (phages) are bacterial viruses and they usually cause lysis of the bacterial cells they infect. They are highly specialized, in that they usually infect only members of a particular species, or even particular strains within a species.

DNA base composition-this base composition is usually expressed as the percentage of guanine plus cytosine (G + C). The base composition of a single species is theoretically a fixed property; thus, a comparison of the G + C content in different species can reveal the degree of species relatedness.

DNA fingerprinting-used to determine the source of hospital-acquired infections. In one hospital, patients undergoing coronary-bypass surgery developed infections caused by Rhodococcus bronchialis. The DNA fingerprints of the patients' bacteria and the bacteria of one nurse were identical. The hospital was thus able to break the chain of transmission of this infection by encouraging this nurse to use aseptic technique.

PCR-(polymerase chain reaction) used to increase the amount of microbial DNA to levels that can be tested by gel electrophoresis

Dichotomous keys are widely used for identification. Identification is based on successive questions, and each question has two possible answers (dichotomous means cut in two). After answering one question, the investigator is directed to another question until an organism is id'd.

• aka phylogeny
• the study of the evolutionary history of organisms

• Systematics examines evolution of biological diversity, and combines data from the following areas:
• -fossil record
• -comparative homologies. Similarity of structures due to shared ancestry
• -comparative sequencing (or reading) of DNA/RNA among organisms
• 
• This tree (or any tree you use to show relatedness between something) is also called a Cladogram

the system of having two names (genus [like our last name] and species [like our first name]) for each organism; also called scientific nomenclature

• First proposed in the 1700s by Swedish scientist Carolus Linnaeus
• Provides a "universal language" for scientists to use when classifying organisms (That universal language is Latin)
• Genus is always capitalized; species are lower case
• Genus and species names are always italicized or underlined when written

• plural genera
• the first name of the scientific name (binomial); the taxon between family and species
• always capitalized and always a noun

the discovery of three cell types was based on the observations that ribosomes are not the same in all cells. Ribosomes provide a method of comparing cells bc ribosomes are present in all cells. Comparing the sequences of nucleotides in ribosomal RNA from different kinds of cells shows that there are three distinctly different cell groups: the eukaryotes and two different types of prokaryotes-the bacteria and the archaea

• a group of closely related organisms that can interbreed
• animals, plants, fungi and protists

a population of cells that share certain rRNA sequences; in conventional biochemical testing, it is a population of cells with similar characteristics

a group of viruses sharing the same genetic info and ecological niche

genetically different cells within a clone

a population of cells arising from a single parent cell

a dichotomous phylogentic tree that branches repeatedly, suggesting the classification of organisms based on the time sequence in which evolutionary branches arose

• DNA base composition
• -Guanine + cytosine
• moles% (GC)
• so just taking all their DNA and seeing how
• many GC% they have in them

Nucleic acid hybridization

DNA fingerprinting-two ways:

-electrophoresis of restriction enzyme digests

-Polmerase chain reaction (PCR)

Analysis of DNA by electrophoresis of restriction enzyme fragments of the DNA

• •DNA fingerprinting is used in several ways:
• –paternity tests

–evidence in criminal cases

–immigration requests

–studying biodiversity

– tracking genetically modified crops

Which technique you use depends on how much sample you have

RFLP testing is when you have more than 1000 cells worth of DNA - these are the ones usually used in paternaty testing

PCR Analysis is used when you have more then 20 cells- used in crime situations (ex. skin cells found under fingernails)

• Restriction fragment length polymorphism
• used when there are a lot of cells
• a fragement resulting from restriction-enzyme digestion of DNA

•DNA fingerprints are based on specific sequences of an individual’s DNA

• –Use noncoding regions have repeating DNA
• sequences

–Number of repeats differs between people (different people have different numbers/different species have different numbers)

• –Use restriction enzymes to cut the repeats
• –Different numbers of repeats are different “sizes”
• –When cut DNA is run out on a gel by size, different patterns emerge
• –Banding pattern on a gel is a DNA fingerprint

• A type of DNA fingerprinting used when we have very few cells
• Polymerase chain reaction a technique using DNA polymerase to make multiple copies of a DNA template in vitro (since we have so little we need to replicate the cells)

• Purpose
• – Quickly make many copies of a region of a DNA
• molecule (eg. The area of unique DNA repeats)

• Method
• – Multiple rounds of DNA replication

• Components in PCR reaction
• – Target DNA, nucleotides, DNA polymerase, and primers

Temperature cycling – DNA replication controlled by temperature

(be sure to watch the PCR video she included under CH10)

• Temperature cycling –
• PCR process uses a machine (thermocycler) in
• which PCR reaction goes through ~30 cycles of three different temperature changes:

~95ºC – Melting temperature – separates double stranded DNA

50-65ºC – Annealing temperature – allows primers to bind to DNA antisense strand

72ºC – Extension temperature – allows DNA polymerase to copy the DNA region between the primer regions

AN EXPONENTIAL INCREASE IN THE NUMBER OF COPIES OF THE TARGET DNA REGION(S)!

• PCR analysis
• DNA is synthesized using Taq polymerase

the separation of substances (such as serum proteins or DNA) by their rate of movement thru an electrical field

An enzyme that cuts double-stranded DNA at specific sites between nucleotides

Polymerase chain reaction (PCR) analysis

• DNA is synthesized using Taq polymerase (from Thermus aquaticus)
• Archaea bacterium from the Yellowstone hot
• springs

Taq polymerase does not denature at high temperatures (like ours does)

• Allowed for the genetic revolution in biology because now we can make copies of DNA of any kind of organism so we could study it
• We makes enough copies of it so we could put it on a gel or an expensive machine that will read the DNA sequence back to you

Enzyme Linked Immunosorbent Assay

• · Technique
• used to detect (assay) specific molecules (e.g. proteins & carbohydrates)
• in samples.

• · Immunological
• technique: uses antibodies and antigens.

· Quantitative.

· Very sensitive.


· Commonly used in medicine and scientific research

• •Combine known antibody plus unknown
• bacterium/virus-We take an antibody then you see if there is a response to the foreign bacteria or viruses

• •Slide agglutination (clumping)
• 
• positive means the antibody reacts to the bacteria/virus

• •ELISA- Enzyme Linked Immunosorbent Assay
• Technique used to detect (assay) specific molecules (e.g. proteins and carbohydrates made by bacterias or viruses) in samples
• Immunological technique: uses antibodies and antigens
• quantitative (measurable)
• very sensitive (so we dont need a large sample..a little bit is enough)
• commonly used in medicine and scientific research

• also used for pregnancy tests
• looks for HCG (human chorionic gonadotropin) - these are present if you are pregnant

Gram Negative Bacteria

HUGE group that includes a wide variety of pathogens, such as Escherichia, Salmonella, Vibrio, Helicobacter, and many other notable genera.

Others are free-living, and include many of the bacteria responsible for nitrogen fixation.

Group defined primarily in terms of ribosomal RNA (rRNA) sequences.

Named for the Greek god Proteus, who could change his shape, because of the great diversity of forms found in this group.

Divided into five sections, referred to by the Greek letters alpha through epsilon, again based on rRNA sequences.

the list of Proteobacteria:

• Alphaproteobacteria
• Betaproteobacteria
• Neisseria gonorrhoeae
• Gammaproteobacteria
• Escherichia coli
• Salmonella pullorum
• Vibrio cholerae
• Haemophilus influenzae
• Deltaproteobacteria
• Epsilonproteobacteria
• Helicobacter
• pylori

Low G&C Gram Positive

• List of Firmicute:
• Clostridium
• C. tetani
• Mycoplasmas
• Bacillus
• B. anthracis
• Lactobacillus (as shown under this list)
• Streptococcus
• S. pneumoniae
• S. pyogenes
• Staphylococcus
• S. aureus
• S.epidermidis

• Phylum Firmicutes
• Lactobacillus

• Rod-shaped fermenters. Facultative anaerobic or microaerophilic bacteria.
• They are usually straight, but can sometimes form spiral or coccobacillary forms. Often found in pairs or chains of varying length.
• Produce lactic acid. Used in dairy product production.
• Required for the maintenance of healthy intestinal microflora.
• 

D. a supply of the four DNA nucleotides

Components in PCR reaction–
Target DNA, nucleotides, DNA polymerase, and primers

a. restriction enzymes this happens during the melting phase
b. complementary sequences of RNA this happens during the reaction using the taq polymerase
c. large amounts of DNA cannot be this because PCR uses small amounds of DNA

d. a taxon comprised of classes

• Remember
• King Kingdom
• Philip Phylum
• Came Class
• Over Order
• For Family
• Green Genus
• Soup Species

• a. a taxon composed of families Genus is composed of families
• b. a taxon comprised of one or more orders and below kingdom It IS below kingdom but is not comprised of one or more orders. It contains one or more CLASSES
• c. a taxon belonging to a class contains classes, doesn't belong

C. Indirect ELISA

ELISA is used to test proteins and carbohydrates that are made by bacterias or viruses including HIV virus and in pregnancy tests

a. DNA fingerprinting is used for paternatiy testing, criminal cases, studying biodiversity, etc
c. Biochemical testing used to separate species of bacteria into distinct species; not ideal to find presence of HIV
d. Cytopathic effects are the structural changes in a host cell resulting from viral infection. She didn't go over this and it cant be the answer since its an effect and not a test

D. systematics

a. nomenclature-is the name
b. identification-finding out what belongs in which class (determinative)
c. systematics-the study of the evolutionary history of an organism
d. classification- list of characteristics of why there belong where they are

D. actinobacteria

since it lacks a nucleus and has a thick peptidoglycan wall, we must look for a Gram Pos prokaryote

a. plantae -not a prokaryote
b. chlorflexi -this is gram Neg
d. proteobacteria -also gram Neg

D. Bacillus subtilis

Genus is always capitalized; species are lower case
Genus and species names are always italicized or underlined when written

B. multiple noncoding DNA segments

as used by RFLP for the DNA fingerprinting

D. all make cysts

remember that protozoa (from past chapters) are from the Eukarya domains, so they have eukaryotic cells, may produce sexually, and may have flagella or cilia

Enzyme

Binomial Nominclature

restricting

cladogram

hyphae