Micro F Microbial Genetics

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bacterial viruses

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bacteriophage

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Important properties of Bacteria

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1) Small (~1microm)
2)Haploid genome
3)Rapid Growth
4)May be grown in large #s
5)May be separated and grows into colonies
6)can isolate and determine # of viable bacteria by dilution and plating
7) by defining nutrients in agar one can a) id nutrient requirements b)select cells with specific nutrient requirements

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By defining the nutrients in the agar of a bacterial culture, one can:
a) ID (what)?
b) select for (what)?

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a) ID nutritional requirements (i.e. biosynthetic capabilities
b) Select cells with specific nutritional requirements

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describe the bacterial genome

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a single
double-stranded
circular
DNA molecule
(example: E. coli chromosome ~4000kb pairs, encoding ~4000 genes)

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E. coli chromosome
how big is it?
how many genes does it encode?

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bacterial chromosome that is:
~4000kb pairs long (dsCircDNA)
encoding ~4000 genes

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describe bacterial DNA replication

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SEMICONSERVATIVE (progeny molecules consist of a parental strand and a nascent strand)
BIDIRECTIONAL (originating from a single point called an origin of replication ORI)

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types of bacterial DNA polymerase

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a) one involved in DNA repair (mutants UV &mutagen sensitive)
b) one responsible for chromosomal replication (mutants lethal)

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polymerase involved in replication of bacterial genome

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DNA POL III

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Genotype

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nucleotide sequence of the genome

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mutations in genome are changes in what?

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genotype (can cause changes in phenotype)

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phenotype

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observable characteristics of an organism

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SILENT MUTATION

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mutation that does not give rise to a change in phenotype

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what types of mutations can be SILENT?

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1. if genetic code is REDUNDANT (e.g. GCC, GCA, etc.. all code for alanine)
2. CONSERVATIVE CHANGES in amino acids may not affect function (eg: valine to alanine [both nonpolar])

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Types of mutations

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1. Point mutations
2. Deletion
3. Insertion
4. Frameshift
5. Revertant

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Point mutations

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single base changes
a) TRANSITIONS: purine to purine or pyrimidine to pyrimidine
b) TRANSVERSION: purine replaced by a pyrimidine or vv
may change codon to encode for:
-another amino acid
-a stop codon

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Deletion Mutation
Insertion Mutation

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removal of one or more nucleotides
addition of one or more nucleotides

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Frameshift mutation

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a shift in reading frame
caused by an insertion or deletion of nucleotides

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Revertant mutation

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mutation that restores a WT phenotype
a) true revertants = reversal or original mutation
b) Suppressors = mutations which occur at a second site and restore WT phenotype

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Suppressor Mutation

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occurs at a second site and restores WT phenotype in a mutant genotype

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Direct selection of mutants
(ampicillin resistent cells)

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bacteria grown in liquid

are plated on solid medium (agar plates) containing ampicillin

only apicillin resistant organisms will grow and form colonies

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why is the haploid nature of the bacterial genome KEY in its utility as a genetic tool?

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THE PHENOTYPE CONFERRED BY A RECESSIVE MUTATION IS IMMEDIATELY APPARENT.
(whereas recessive mutations can be masked in diploid eukaryotic cells)

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Auxotrophy

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inability to synthesize an essential metabolite (e.g. an amino acid)

these mutants = auxotrophs
WT (w/ respect to that metabolite) = prototroph

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Minimal medium contains:

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CARBON SOURCE (e.g. glucose)
NITROGEN SOURCE (ammonium sulfate)
PHOSPHATE SOURCE (sodium phosphate)

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What can a WT organism make when grown on MINIMAL MEDIUM?

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WT organism has the biosynthetic capability to make:
ALL OF THE ESSENTIAL METABOLITES when grown on this

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an ARGININE AUXOTROPH will only grow on:

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a minimal medium supplemented with arginine

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a PROTOTROPH will grow on

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MINIMAL MEDIUM (C, N, Pi)

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Easily observable phenotypic changes (in bacterial genetic studies)

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-AUXOTROPHY
- Inability to utilize a particular CARBON SOURCE
-Antibiotic Resistance
-Colony morphology mutants

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Colony morphology mutants

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alteration of membrane components (LPS, flagellin, pili) may alter colony size and shape

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Two mechanisms by which antibiotic resistant strains of bacteria arise:

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a) mutation to antibiotic resistant
b) transfer of antibiotic resistant genes

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In patients treated with an antibiotic, the % of antibiotic resistant organisms is proportional to what?

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LENGTH OF TREATMENT

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What principle underlies the development of antibiotic resistant strains?

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Darwin's NATURAL SELECTION
-mutations appear spontaneously
-antibiotic treatment = selective pressure
-antibiotic resistant organisms survive, become principle component of new population

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What did the LEDERBERG EXPERIMENT show?

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natural selection = the basis of antibiotic resistance
1)resistant mutations appeared spontaneously PRIOR to antibiotic exposure
2) resistance is due to SELECTION of mutants (w/ existing resistance to antibiotics)

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What can a CROSS-FEEDING ASSAY be used for?

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to order the genes in a biosynthetic pathway
(using auxotrophs for the different genes)

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bacterial translation is accomplished by what?

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the 70s RIBOSOME

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What is required for bacterial translation?

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70S Ribosome
Shine Delgarno Box (upstream from AUG)
Initiation Codon (AUG) =met?
aminoacyl tRNA
stop codon

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Transition Mutation

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point mutation
purine --> purine (A-->G)
or
pyrimidine --> pyrimidine (C-->T)

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Tranversion Mutation

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point mutation
purine <--> pyrimidine
(A-->C) or (G-->T)

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Cross Feeding Assay???

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F-6

somehwat counter-intuitive, as auxotrophs for mutations early in pathway, grow more than those with downstream mutations (cause downstream products are supplied by other auxotrophs, and they can complete rest of pathway)

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How can auxotrophic mutants be isolated using Penicillin?

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-Auxotrophic mutants will not grow in a minimal medium (+ Penicillin)
-Prototrophs will grow AND be Killed (Penicillin only kills growing cells, can't build cell wall)

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CONDITIONAL MUTATIONS

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exhibit a mutant phenotype only under certain conditions (e.g. temp sensitive {ts} mutations)

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TS mutations in E. coli

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E coli grows optimally at 37 degrees C
1) certain mutations silent at 30C (PERMISSIVE TEMP)
2) mutant phenotype is observed at 42 C (NONPERMISSIVE TEMP)

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what is observed at the Nonpermissive Temperature in TS mutations?

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the MUTANT PHENOTYPE is observed

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A TS Arginine Auxotroph
a) will not grow:
b) will grow with:

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a) will NOT grow at 42 C on min. medium (w/o arginine)
b) WILL grow at 30 C both with and WITHOUT ARGININE!

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Selection of temperature sensitive mutations permits isolation of what?

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mutations in essential genes

eg: DNA POLYMERASE mutants

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molecular basis of conditional mutations

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protein can assume normal conformation at PERMISSIVE TEMP

while at higher nonpermissive temp the protein fold abnormally and will not function

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Types of genetic recombination

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a) General recombination (requires extensive homology)
b) Site-specific recombination (requires small region of homology)
c) Illegitimate recombination (requires no homology)

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GENERAL RECOMBINATION

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requires extensive homology between recombining DNA molecules

protein recA helps mediate (catalyzes base pairing bt donor and recipient)

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SITE-SPECIFIC RECOMBINATION

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requires limited homology
e.g. site-specific integration of a lysogenic bacteriophage such as λ.

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ILLEGITIMATE RECOMBINATION

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no homology exists between recombining DNA molecules.

e.g. Random integration of transposons and insertion sequences.

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Use of COMPLEMENTATION to elucidate biosynthetic pathway

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if one has isolated series of mutant with same phenotype, can be used to determine the # of independent genetic elements

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Genetic Complementation Test

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create partial diploids by coinfecting cells with 2 mutants (both lethal to K12 E.Coli for ex)
if mutants COMPLEMENT and phage multiplication occurs, then mutations are in different genes

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bacteriophage

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bacterial viruses
consist of a nucleic acid genome (DNA or RNA) + protein coat (shell or capsid)
small (10 genes) or large (>150 genes)

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bacteriophage morphology

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simple (helical or icosahedral nucleocapsid)
or
complex (head, tail and tail fibers)
{tailless, tailed, filamentous}

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what determines the species of bacteria a particular phage infects?

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determined by the viral coat of proteins on the phage

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how are transpoable genetic elements different from bacteriophage?

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have the property of self-transmissibility

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How are bacteriophage grown?

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1. infect actively growing bacterial culture, bacteriopage reproduce, and in most cases, cause host cell lysis w/ release of progeny phage (centrifuged, supernatant removed)
2. Bacteriophage left, quantified by plaque assay on bacterial lawn: plaques represent zone of bacterial death (each contains several million phage)

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Plaque Assay

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way to quantify bacteriophage
1) phage placed on lawn of sensitive bacteria
2) their growth (infection of bacterium, multiplication, release by bacterial lysis, reinfection of adjacent bacteria) produces plaque (zone of bacterial death, containing millions of phage)

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types of phage lifecycles

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lytic
and
lysogenic

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lytic pathway

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involves
-phage multiplication
-release of newly formed phage following host cell lysis
(virulent phage can only follow the lytic pathway)

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lysogenic pathway

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phage lifecycle that does not result in the production of progeny phage or bacterial killing

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life-cycle pathway of temperate phage

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can follow lytic or lysogenic pathway

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Steps in life cycle of a virulent DNA phage

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1. Adsorption (phage coat protein binds to cell membrane receptors)
2. Intro of DNA (nucleic acid goes in, coat protein stay out)
3. Transcription of phage DNA and inhibition of Host transcirption
4. Replication of viral DNA
5. Synthesis of phage capsid proteins
6. Morphogenesis and packaging of phage genomes
7. Lysis and release

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One-Step growth curve
-represents reproduction of what?
-what does it indicate?

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-pattern of phage reproduction
-indicates that phage do not reproduce by binary fission (only page nucleic acid enters the cell, viral components are syntehsized and then assembled into mature phage particles late in infection)

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virulent phage produce what?

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produce CLEAR PLAQUES
they infect cells, replicate, lyse the host, infect more cells

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temperate phage produce what kind of plaques?

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produce TURBID PLAQUES
because the temperate phage can either cause:
a)VIRULENT (lytic) infection (occurs in most cells) or
b)LYSOGENIC INFECTION- does not result in progeny phage and bacterial killing, rather phage DNA becomes integrated in the host chromosome, is then propagated as a prophage when the bacteria divide

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Bacteriophage Lambdis is what type of phage?

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well characterized TEMPERATE PHAGE (following injection of its DNA, makes choice to undergo a lytic or lysogenic infection)

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what happens when a phage decides to undergo lysogenic infection?

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-following injection of DNA chromosome circularizes (it was linear in virion)
-SITE SPECIFIC INTEGRATION into bacterial chromosome
-lamda repressor, prevents lytic infection

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integrated phage DNA is called

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a prophage

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how is the prophage successfully carried by the E. coli chromosome

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expresses a REPRESSOR of all the genes required for lytic infection

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effect of stress on prophage?
starvation, UV damage

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lambda repressor is inactivated
prophage excises and undergoes a virulent infection

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How do bacteria protect themselves from bacteriophage infection?

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RESTRICTION ENZYMES (degrade infecting DNA)
MODIFYING ENZYMES (chemically modify self DNA to protect it from restriction enzymes)

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restriction enzyme EcoR1

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restricton enzyme
recognizes palandromic sequence
GAATTC
CTTAAG
digests it to:
G AATTC
CTTAA G

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modification enzyme Eco R1 methylase

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chemically modifies the EcoR1 restriction by methylating adenine (adds CH3 group) to protect it from EcoR1 restriction Enxymae

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Transposons

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-mobile genetic elements that are integrated into bacterial chromosomes or plasmids, capable of jumping from one location in DNA to another
-Insertion Sequences (IS) on ends allow ILLEGITIMATE RECOMBINATION
-ISs alone = simplest transposons

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Transposition requires what enzyme?

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a transposase enzyme encoded by the IS element (that allows it to jump)

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Typical transposons consist of:

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a gene encoding antibiotic resistance flanked by IS elements

some carry toxin genes

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transposons that encode antibiotic resistance can dublicate during transpotion so that they are retained at their original site, this can result in...

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movement of antibiotic resistance gene from bacterial chromosome to a conjugal plasmid and the reverse

important role in spread of antibiotic resistance among pathogens
?????? F-17

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3 known mechanisms of GENE TRANSFER in bacteria:

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a) TRANSFORMATION (naked DNA from one bacterium to another)
b) CONJUGATION (sexual reproduction, mediated by F plasmids)
c) TRANSDUCTION (tranfer of a gene from one bacterium to another by a phage that has replaced part/all of its genome with some of its' hosts DNA)

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two types of TRANSDUCTION

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GENERALIZED
and
SPECIALIZED

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forms of gene transfer used as genetic tools to manipulate and map bacterial genes

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TRANSORMATION
and
TRANSDUCTION

(not so much conjugation)

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TRANSFORMATION

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1) Important historical role: first evidence DNA = genetic material
2)observed in both gram(+)&(-) bacteria
3) DNA binds to bacterial membrane, enters cell and undergoes HOMOLOGOUS RECOMBINATION with host chromosome
4) Main value: easily introduces recombinant DNA plasmids into bacterial strains
5) competence = ability of bacteria to be transformed

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COMPETENCE

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ability of bacteria to be transformed (take up naked DNA via transformation)

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How can E. coli be made competent?

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by treatment with CaCl2 or by ELECTROPORATION
(USED WIDELY IN MOLECULAR CLONING)
makes E. coli competent (can take up naked DNA)

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generalized transduction is accomplished by what group?

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a unique group of bacteriophage that produce some pahge particles that contain only host DNA (Bacteriophage P1)

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generalized transduction is used extensively for what type of studies?

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MAPPING STUDIES

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generalized transduction results from what mistaken process?

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the mistaken encapsidation of host chromosomal DNA fragments into phage particles

host DNA can be from any part of the bacterial chromosome

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how is generalized transduction accomplished in the lab?

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phage preparation grown on a donor strain of bacteria is used to infect a recipient strain of bacteria

donor DNA present in transducing phage particles will recombine with the bacterial chromosome of the recipient strain by general recombination

recipient cells can be selected that have acquired genetic markers present in the donor strain

(ONLY CLOSELY LINKED MARKERS ARE TRANDUCED)

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MAPPING BY GENERALIZED TRANSDUCTION
(F-19)

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donor phage P1 stock grown on E. coli strain prototrophic for Leucine, arabinose, threonine
-used to infect an E. Coli strain auxotrophic for all 3 markers
-WT allele is selected
-Frequentcy of acquisition of unselected alleles is measured (recombination ~ proportional to linkage on chromosome)

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Specialized transduction

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temperate phage integrate into host cell chromosome at a specific location
-rarely during transition from lysogeny to veg. growth, prophage excises incorrectly leaving behind some of its' own gene and acquiring a limited set of bacterial genes on either side of attachment site
-can transfer these bacterial genes to another bacterium (only genes near site of integration in the bacterial chromosome will be transduced)

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specialized transducing phage

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contains both DNA derived from the host bacterium and the phage (due to incorrect excision of prophage (with some phage genes excised and a few bacterial genes acquired on either side of attachment site)

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only genes near what will be transduced in specialized tranduction?

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only genes near the site of integration in the bacterial chromosome

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plasmids

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extrachromosomal
circular DNA molecules
capable of autonomous replication

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first type of plasmid discovered

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F factor

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how was F factor plasmid identified?

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its ability to transfer chromosomal genes from a donor straingbearing and F factor (F+) to a recipient (F-) strain

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mating in bacteria is called

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conjugation

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all plasmids have an origin of DNA replication and frequently carry optional genes whic confer additional phenotypic properties on their host bacteria, such as:

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-fertility
-resistance to: antibiotics, metals, uv-irradiation, bacteriophage
-production of: bacteriocins, proteases, toxins, antigens, hemolysins

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most medically important class of plasmids

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R Factors (encode resistance to various antibiotics and are an important cause of hospital acquired infections)
R Factors are CONJUGATIVE

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bacteriocidins

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class of plasmids, encode antibiotics called BACTERIOCINS

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bacteriocins

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antibiotics encoded by BACTERIOCIDINS (class of plasmids)

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charateristics of plasmids

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1. circular, supercoiled, dsDNA
2. range in size from 1.5kb-400kb pairs (F&R plasmids are large)
3. can be physically separated from chromosomal DNA by density centrifugation or agarose gel electrophoresis
4. plasmid are capable of autonomous replication because they have an origin of DNA replication (replication & transcription machinary, provided by host enzymes)

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Copy Number of plasmids

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# of plasmid "copies" present per chromosome
-# is regulated (by plasmid encoded repressor of replication)
-certain plasmids are present in just 1 copy, others present at 10-50 copies per chromosome

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classification of plasmids can be made according to what kind of groups?

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INCOMPATABILITY GROUPS
-classification of plasmids based on their inability to coexist in the same cell
PHYSICAL MAP
-classification of plasmids based on similarity of endonuclease digestion pattern

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INCOMPATABILITY GROUPS classify similar plasmids because

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-similar plasmids share the same repressor mechanism controlling copy #, cannot exist stabley in same hosts (usually share extensive DNA homology and form an incompatability group)

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RESTRICTION ENDONUCLEASE MAP can be used to classify plasmids

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similarities in endonuclease restriction patterns indicate regions of homology

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Plasmids frequently contain IS elements (transposons)

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-side effect = genomic rearrangements, therefore play role in plasmid evolution
-in F plasmids, IS elements allow integration into host chromosome to form an Hfr strain
-IS elements flanking antibiotic resistance genes in R factors are responsible of multiply resistant R factors

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Hfr strain

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a bacterium with a conjugative plasmid (often F) integrated into its genomic DNA. Hfr is the abbreviation for high frequency recombination

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what is responsible for the generation of multiply resistant R factors

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IS elements (transposons) flanking antibiotic resistance genes in R factors

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conjugation

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sexual reproduction in bacteria
-exchange of genetic info bt two parents is unequal
-DNA transferred unidirectionally bt donor strain (F+) and a recipient (F-) strain
-transfer of DNA requires cell to cell contact

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conjugation was discovered by:

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LEDERBERG (while a med student)
mixed A+B+C-D- lac+ strain w/ A-B-C+D+ lac- strain
incubated, then plated on A-B-C-D- medium and got A+B+C+D+ and lac- progeny
colonies had unselected markers from one recipient parent (indicating unidirectional transfer, male to female)

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F plasmid encodes for proteins required for what?

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proteins required for conjugal transfer
-SEX PILUS = conjugation bridge, made of pilin (protein)
-proteins required for transfer of tra genes (a transfer origin, nicked to initiate conjucation)

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an F+ strain will only initiate pilus formation with what?

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an F-strain

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What does it mean when an F+ strain is "cured"

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it has lost its F plasmid under certain growth conditions

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HFR strain formation

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1) Extrachromosomal F plasmid occasionally integrates into bacterial chromosome (1 in 10^4 cells)
2) Both plasmids and bacterial chromosomes have multiple IS elements (integration occurs via homologous recombination bt IS elements)
-cell with an integrated F+ cell = HFr cell

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F' plasmid

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HYBRID PLASMID
formed by impercise excision of F from Hfr cell (carrying nearby chromosomal genes from Hfr)

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Mechanism of Hfr transfer

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when Hfr mates with an F- cell, Hfr can transfer donor chromosomal genes to the F- cell
1) PILUS FORMATION,
2) NICKING of tra and strand transfer, 3) DNA REPLICATION
4) PILUS RUPTURE
5) HOMOLOGOUS RECOMBINATION

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MAPPING BY INTERRUPTED MATING
by physically breaking pillus, showed that:

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1. genes transfered in a fixed order
2. genes near ori are transferred more efficiently near the ori T
3. were able to generate map of entire E. coli chromosome

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resistance transfer factors

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1) 1st discovered in strain of enteric bacteria in Japan, resistant to several different antibiotics
2) emergence of multiply resistant strains of pathogenic bacter = medically significant
3. increase has correlated with increasing use of antibiotics in the treatment of infectious disease and as a supplement in livestock feed
5. Many RTFs capable of interspecies transfer
6. In most cases resistance genes are flanked by IS sequences (can be transmitted from one R- factor to another by transposition---> how multiple resistance arises)
7. frequent treatment with subinhibitory dose of an antibiotic often causes the emergence of cells with high resistance (in these cells, the RTF contains multiple copies of the resistance determinant that have arisen by gene duplication.)