Microbiology Exam 2

Diversity in bacteria can be generated via

Mutation

Mutation

–Modification in the sequence of DNA in a gene often resulting in an alteration in the protein encoded by the gene

•Spontaneous

•Induced

Essential for understanding genetics

Spontaneous Mutation

•Mutations are stable inheritable changes in the base sequence of DNA

Vertical Gene Transfer

Mutation passed onto progeny; inherited

Reversion

Change in a cell’s genotype and phenotype to its original state through a change in the mutated gene

Spontaneous mutations can occur as results of

•Base substitutions

•Removal or addition of nucleotides

•Transposable elements

Base substitutions

–Most common type of mutation

–Results from mistakes during DNA synthesis

Incorrect base is incorporated into DNA

Point Mutations

Missense Mutation

Nonsense Mutation

Null or knockout mutation

Point mutations

Occur when one base pair is changed

Missense mutation

Mutation resulting from amino acid substitution

Nonsense mutation

Mutation that changes an amino acid codon to a stop codon

Null or knockout mutation

Mutation that inactivates a gene

Removal and addition of nucleotides

–Shifts the translational reading frame

•Shifts the codons

Frameshift Mutation

•Affects all amino acids downstream from addition or deletion

–Mutations frequently result in premature stop codons

Induced Mutations

–Mutations can be intentionally produced to demonstrate function of particular gene or set of genes

•Mutations can be induced via

–Chemical mutagens

–Transposition

–Radiation

•Chemical mutagens

–Chemical modification of purines and pyrimidines

•Increase frequency of mutations as DNA replicates (base substitutions)

–Nitrous acid

–Alkylating agents

–Base analogs

•Chemicals that are structurally similar to the nitrogenous bases but have slightly altered base pairing properties (base substitutions)

–May result in pairing with wrong base as complementary strand is being syn.

–Intercalating agents

Chemical Mutagen

•Molecules that insert themselves between adjacent bases

•Increase the frequency of frameshift mutations

–Create space between bases

»Extra base is often added to fill space

Ethidium Bromide

Common Intercalating Agent

–Potential carcinogen

–Used to stain DNA in gel electrophoresis

•Transposition

–Common procedure used to induce mutation in laboratory

–Transposon (transposable element)

•Genes that move from one replicon to another site in the same replicon, or to another replicon on the same cell

Insertion Mutation

–Gene that receives the transposon will undergo a knockout mutation

•Transposable elements

–Special segments of DNA that move spontaneously from gene to gene

•Elements called transposons

–Transposons disrupt proper function of gene

•Gene or gene product generally non-functional

Two types of Radiation

Ultraviolet Light and X rays

•Ultraviolet light

–Causes covalent bonding between adjacent thymine bases

»Forms thymine dimers which distorts DNA

»Prevents replication past the dimer (SOS repair system results in the incorporation of the wrong bases)

•X rays

–Causes single and double stranded breaks in DNA

»Breaks that occur on both strands are often lethal

Major problem in induced mutation

identifying bacteria with desired mutation

–Direct selection

•Involves inoculating population of bacteria on medium on which only mutants will grow

–Used to select antimicrobial resistant or auxotrophic mutants reverted to prototrophic organisms

•Indirect selection

–Required to isolate organisms that require growth factor that parent strain does not have (auxotrophic mutants)

•Replica plating

•Testing for cancer causing chemicals (carcinogens)

–Many mutagens are also carcinogens

–Microbes used to test potential carcinogenic activity

•Tests are based on effect chemical has on microbial DNA

Ames test

•common chemical carcinogen test

–Test assumes that the frequency of reversions is increased by mutagens and that most carcinogens are mutagens

–Tests rate of reversion of Salmonella auxotroph

–Also tests potential lethality

•Horizontal gene transfer

–Genes transferred from one cell to another

•Genes are naturally transferred between bacteria using three mechanisms

–DNA-mediated transformation

–Transduction

–Conjugation

bacterial virus

bacteriophage

•DNA-mediated transformation

•DNA transferred as “naked DNA”

–Involves the transfer of naked DNA from the environment to the recipient cell

•Cells rupture during the stationary and death phase

–The chromosome breaks into small pieces and explodes through the ruptured cell wall

–Recipient “competent” cell picks up piece of the naked DNA

–The naked DNA is integrated onto the recipient  chromosome

•replaces the homologous gene on the chromosome of the recipient cell

–Natural transformation occurs when bacterial cells are “competent”

•bacterial cells are capable of taking up and integrating larger fragments of DNA

•Natural transformation occurs in four stages

–Entry of the DNA

–Integration of the donor DNA

–Mismatch repair

–Cell multiplication

–Entry of the DNA

•Only single strands enter, double strands are degraded

–Integration of the donor DNA

•Donor DNA is integrated by hydrogen bonding

•Enzymes cleave recipient DNA

•Donor DNA is put in place

–Mismatch repair

•Repair mechanism removes either donor or recipient DNA that doesn’t match

•Repairs with correct nucleotides

–Cell multiplication

•Transformed cells multiply under selective conditions in which non-transformed cells will not grow

Other methods of transformation

chemically induced transformation and the use of biolistic transformation (gene guns)

•Transduction

–Bacterial DNA that is transferred from donor to recipient via a bacterial virus (bacteriophage)

–Two types of transduction

•Generalized

–Any gene of donor can be transferred

•Specialized

–Only specific genes can be transferred

•is a mis-packaging of DNA during viral replication

–The mis-packaged  phage infects a new bacterial cell and inserts the donor DNA into the recipient cell

–The donor DNA is integrated into the cell by homologous recombination

Conjugation

•only form of gene exchange in which donor survives

–Conjugation is frequently mediated by a plasmid

•Plasmid is self replicating extrachromosomal piece of DNA

–Can code for traits that give bacteria advantage

–Conjugation requires direct contact between cells

–Cells must be of opposite mating types

•R plasmids

–Group of plasmids that confer resistance to many antimicrobial agents

•Self-transmissible

–Carry all of the genetic information they need for transfer

•Mobilizable

–Encode some but not all of the information needed for transfer

•Example

–F (fertility) plasmid

–Self-transmissible plasmid

•During conjugation, the plasmid is replicated in the donor cell and is transferred to the recipient

–After, plasmid is transferred, F- cell becomes F+

•Some cells can be cured

–Spontaneously lose their plasmid

Basic components of molecular biologist’s “toolkit” include

–Restriction enzymes

–Gel electrophoresis

–DNA probes

–primers

•Restriction enzymes

–Naturally occurring enzymes that cut DNA into fragments

•Cut in predictable and controllable manner

–Generate pieces of DNA called restriction fragments

•These fragments can be joined to new fragments

–Enzymes produce jagged cuts called sticky ends or blunt cuts called blunt ends

»Ends anneal together to form new strand

•DNA ligase covalently joins fragments

•Gel electrophoresis

–Used to separate DNA fragments according to size

•DNA is put into wells in gel

•Gel subjected to current (negative to positive)

•DNA moves through the gel towards positive electrode

–Fragments are separated according to size

»Large fragments remain high in the gel

»Small fragments migrate lower

–Gel must be stained to view DNA

•Oftentimes stained with ethidium bromide solution

•DNA probes

–Used to locate nucleotide sequences in DNA or RNA

–Probe is single-stranded piece of DNA tagged with detectable marker

•Location can be easily determined

–Probe will hybridize to complementary fragment of interest

technologies that employ DNA probes

–Colony blotting

–Southern blotting

–Fluorescence in situ hybridization (FISH)

–DNA/RNA microarrays

Colony blotting

–Used to detect specific DNA sequences in colonies grown in agar plates

•Colonies are transferred in place on nylon membrane

–Colony blots are used to determine which cells contain gene of interest

Southern blotting

–Uses probes to detect DNA sequences in restriction fragments separated using gel electrophoresis

–Application of Southern blotting is locating DNA sequences similar to ones being studied

–Northern Blot – RNA

–Western Blot - Protein

Fluorescence in situ hybridization (FISH)

–Uses fluorescently labeled probe to detect certain nucleotide sequences

•Detects sequences inside intact cell

–Specimen is viewed using fluorescence microscope

–FISH can be used to identify specific properties of bacteria

•DNA microarray technologies

–Used to study gene expression under certain conditions

–DNA arrays are solid supports with fixed patterns of different single-stranded DNA fragments attached

–Entire DNA specimen to be studied is labeled

–Enable researchers to screen sample for numerous sequences simultaneously

Applications for
DNA Sequencing

•Knowing DNA sequence of particular cell helps identify genetic alterations

–Alterations that may result in disease

•Sickle cell anemia

–Due to single base-pair change in gene

•Cystic fibrosis

–Caused by three base-pair deletion

•DNA sequence analysis assists in studying evolutionary relatedness

Dideoxychain termination

–Elements for termination reaction include

•Single stranded DNA template

•Primer that anneal to template

•DNA polymerase

•Each of the nucleotide bases

–One of these bases is labeled with marker for detection

•Dideoxynucleotides

–Like deoxynucleotide counterparts but lack 3’ OH

»Incorporation causes chain termination

–Polyacrylamide gel electrophoresis used to separate DNA fragments by size

Automated DNA sequencing

–Most automated systems use fluorescent dyes to detect newly synthesized DNA

–Gel electrophoresis used to separate fragments into colored bands

–Laser used to detect color differences

•Order of color reflects nucleotide sequence

Primers

–Single-stranded DNA fragments bind the sequences of DNA

–Used in in vitro DNA synthesis

•Primer serves as fragment for addition of DNA nucleotides (PCR)

Polymerase Chain Reaction

•Creates millions of copies of given region of DNA in matter of hours

–Technique exploits specificity of primers

•Allows for selective replication of chosen regions

–Termed target DNA

•Large amounts of DNA can be produced from very small sample

•Starting with double-stranded DNA molecule, process involves number of amplification cycles

•DNA is amplified exponentially

PCR requires three step amplification cycle

–Step 1: double-stranded DNA denatured by heat

–Step 2: primers anneal to complementary sequence of target DNA and DNA synthesis occurs with heat stable DNA polymerase

–Step 3: duplication of target DNA

DNA cloning

–Process of producing copies of DNA

•Cloned DNA generally combined with carrier molecule called cloning vector

–Insures replication of target DNA

Researching gene function and regulation

–Gene expression, regulation and function can be studied by gene fusion

•Joining gene being studied to reporter gene

–Reporter gene encodes observable trait

»Trait makes it possible to determine the conditions that affect gene activity

Why are living organisms divided into groups

To better understand relationships among species

Taxonomy

The science that studies organisms to order and arrange them

Taxonomy can be viewed in three areas

–Identification

•Process of characterizing in order to group them

–Classification

•Arranging organisms into similar or related groups

–Nomenclature

•System of assigning names

True or False

Phenotype can be used in the process of identification of bacteria

True

Methods used to identify prokaryotes

–Microscopic morphology

–Metabolic capabilities

–Serology

Strategies Used to Classify Prokaryotes

–Understanding organisms’ phylogeny assists in classification

•Allows for organized classification of newly recognized organisms

–Development of molecular techniques for classification and identification make genetic relatedness possible

•Taxonomic hierarchies

–Classification categories arranged in hierarchical order

•Species – group of related isolates or strains

–Most basic unit

•Genus – group of related species

•Family – collection of similar genera

•Order – collection of similar families

•Class – collection of similar orders

•Phylum – collection of similar classes

•Kingdom – collection of similar phyla

•Domain – collection of similar kingdoms

–New taxonomic category

Microscopic morphology

–Important initial step in identification

•Can be used to determine size, shape and staining characteristics

–Size and shape can readily be determined microscopically

–Gram stain differentiate Gram (+) from Gram (–)

•Narrows possible identities of organism

–Special stains

•Identifies unique characteristics of organisms

–Acid fast stain

Metabolic capabilities

–Identification relies heavily on analysis of metabolic capabilities

–Culture characteristics

•Colony morphology can give clues to identity

–Green pigment of Pseudomonas aeruginosa

-b-hemolytic colonies of Streptococcus pyogenes

  (beta=“complete” hemolysis)

Biochemical tests

•More conclusive identification

–Most tests rely on pH indicators or chemical reaction that results in a color change when a compound is degraded

–pH change can be acidic (i.e. fermentation of sugars), alkaline (production of CO₂ which can raise the pH) or no change at all (could be due to the bacteria not growing or utilizing the specific nutrient source)

Catalase test

–Bacteria that produce catalase break down hydrogen peroxide to release oxygen gas causing bubbling

•Also done on plates

•Sugar Fermentation

–Fermentation of sugars results in acid production causing pH indicator to turn yellow (inverted tube w/n larger tube traps any gas produced)

•Urease test

–Breakdown of urea by urease enzyme releases ammonia and CO₂ leads to alkaline environment within tube as indicated by pink color

•Breakdown of urea by urease enzyme releases ammonia and CO2

MACCONKEY AGAR

–identification of lactose fermenting, Gram-negative enteric pathogens (differential)

–inhibiting growth of Gram-positive organisms (selective).

–Fermentation of lactose turns the medium red/pink. (acidic environment from fermenting lactose).

Biochemical typing

–Biochemical tests can be used to identify species

•They can also be used to identify strains by tracing specific biochemical characteristics called biovar or biotype

•Serological typing

–Identification made based on differences in serological molecules (molecules that react with antibodies)

–Serological characteristics are termed serovar or serotype

•Serology

–Technique relying on specific interaction between antibodies and antigens

–Serological tests are available for rapid detection of many organisms

•E.coli O157:H7

Using Genotype to
Identify Prokaryotes

•Nucleic acid probes can locate unique nucleotide  sequence of a particular species

•PCR

–Used to amplify sequences that allow for detection of specific sequences for identification

•Sequencing ribosomal RNA genes

–There is little genetic variation in rRNA

•16S rRNA is “gold standard” for identifying unknown

    bacteria and determining evolutionary relationships (this is how “tree of life” was determined)

•Advantage

–Identification of organism that can’t be grown in culture

•Genomic typing

–Restriction fragment length polymorphisms (RFLPs)

•Uses restriction enzymes to digest DNA from each organism

•Resolved using pulse-field gel electrophoresis

•Variation in fragments b/w organisms are termed polymorphisms

•National Molecular Subtyping Network for Foodborne Disease Surveillance (PulseNet)

–Catalogs RFLPs of certain foodborne pathogens

Antibiograms

–Identify organisms based on antibiotic susceptibility

–Disc impregnated with antimicrobial placed on inoculated plate

•Clear indicates microbial susceptibility

•Different strains will have different susceptibility pattern

True or False

Prokaryotes display an amazing degree of metabolic diversity

True

True or false

Diversity allowed prokaryotes to emerge as first life-forms,

due to radically different environment on earth 4 billion years ago

True

True or false

Prokaryotes continue to thrive in areas inhospitable to eukaryotic life,

e.g. thermal hotsprings, acidic, alkaline, salty environments, areas

with no oxygen, no sunlight, etc.

True

True or false

Metabolic diversity of prokaryotes allows eukaryotes (us!) to live on

earth, some portions of e.g. nitrogen, sulfur cycles only provided

by prokaryotes, large portion of photosynthesis provided by prokaryotes

True

True or false

Considering that mitochondria and chloroplasts are derived from

prokaryotes, all life on earth is due to metabolic diversity

of prokaryotes.

True

Phototroph

harvests energy from sunlight

Photoautotroph

obtains carbon from CO₂

Photoheterotroph

obtains carbon from organic compounds

Restriction enzymes:

A. Recognize specific DNA sequences

B. Cleave DNA

C. Are used to clone DNA

D. All of the above

E. None of the above

D. All of the above

DNA probes are used for all of the following EXCEPT:

A. Southern blot

B. Microarrays

C. Western blot

D. Colony blot

E. fluorescence in situ hybridization (FISH)

C. Western blot

PCR requires all of the following EXCEPT:

A. DNA template

B. RNA primer

C. Thermostable DNA polymerase

D. deoxynucleotides

B. RNA primer

Urease test is useful to identify:

A. Salmonella typhimurium

B. Helicobacter pylori

C. Neisseria gonorrhea

D. Psudomonas aeruginosa

B. Helicobacter pylori

Anoxygenic phototroph

phototroph that does not produce O₂

Oxygenic phototroph

phototroph that produces O₂

Chemotroph

harvests energy by oxidizing chemicals

Chemolithotroph

oxidizes inorganic chemicals

Chemoorganotroph

oxidizes organic chemicals

Aerobic respiration

Uses O₂ as terminal electron acceptor