h igh f requency of r ecombination (hfr)

High Frequency of Recombination(Hfr)

...bacteria exhibiting a high frequency of recombination,

…the F factor is integrated into the chromosomal genome.

F factor and Chromosomal DNA are Transferred

Recombination Requires Crossing over

Double Crossover

Incomplete Transfer of DNA

• Interrupted Mating: a break in the pilus during conjugation stops the transfer of DNA,

• Transfer occurs at a constant rate,

– provides a means to map bacterial genes.

How Do You Interrupt Bacterial Mating

spread on agar

mate for specified time

frappe

Hfr and MappingHfrH

strs (sensitive to streptomycin) thr+ (able to synthesize the amino acid threonine) azir (resistant to sodium azide)tonr (resistant to bacteriophage T1) lac+ (able to grow with lactose as sole source of carbon) gal+ (able to grow with galactose as sole source of carbon)

F-

strr (resistant to streptomycinthr- (threonine auxotroph)azis (sensitive to sodium azide) tons (sensitive to phage T1)lac- (unable to grow on lactose) gal- (unable to grow on galactose)

Hfr and MappingHfrH

strs (sensitive to streptomycin)thr+ (able to synthesize the amino acid threonine)

F-

strr (resistant to streptomycin)thr- (threonine auxotroph)

Streptomycin kills the HfrH cells in the mating mix.

No threonine kills the F- cells in the mating mix.

Hfr and Mapping

HfrH

azir (resistant to sodium azide)tonr (resistant to bacteriophage T1) lac+ (able to grow with lactose as sole source of carbon) gal+ (able to grow with galactose as sole source of carbon)

F-

azis (sensitive to sodium azide) tons (sensitive to phage T1)lac- (unable to grow on lactose) gal- (unable to grow on galactose)

Interrupting Bacterial Mating

spread on selective media

mate 9 min blend

Replica Plating

After 9 minutes, only azide resistant cells grow.

10 Minutes

Azide, and bacteriophage resistant cells grow.

15 Minutes

Azide, and bacteriophage resistant cells, and lactose utilizing cells.

18 Minutes

All recombinants grow.

% c

ells

wi t

h m

arke

rs

Bacterial Map Distances

units = minutes

HfrH

F factor inserts in different regions of the bacterial chromosome,

Also inserts in different orientations.

Replication Origin

Hfr Order of transferstrain

H thr azi ton lac pur gal his gly thi 1 thr thi gly his gal pur lac ton azi 2 lac pur gal his gly thi thr azi ton 3 gal pur lac ton azi thr thi gly his

F factor

Hfr F-

A

a

Indicates direction of transfer.

A

A

a

Hfr DNA that is not incorporated in the F- strand, and DNA that has crossed out of the F- strand is

digested.

F factor

Hfr F-

A

Leading Gene: the first gene transferred is determined empirically.

A

Hfr

A

F-

A

A transfers first.

A transfers last.

Hfr Order of transferstrain

H thr azi ton lac pur gal his gly thi 1 thr thi gly his gal pur lac ton azi 2 lac pur gal his gly thi thr azi ton 3 gal pur lac ton azi thr thi gly his

Microbes……in the news.

Hfr and MappingHfrH

strs (sensitive to streptomycin) thr+ (able to synthesize the amino acid threonine) azir (resistant to sodium azide)tonr (resistant to bacteriophage T1) lac+ (able to grow with lactose as sole source of carbon) gal+ (able to grow with galactose as sole source of carbon)

F-

strr (resistant to streptomycinthr- (threonine auxotroph)azis (sensitive to sodium azide) tons (sensitive to phage T1)lac- (unable to grow on lactose) gal- (unable to grow on galactose)

Hfr and MappingHfrH

strs (sensitive to streptomycin)thr+ (able to synthesize the amino acid threonine)

F-

strr (resistant to streptomycin)thr- (threonine auxotroph)

Streptomycin kills the HfrH cells in the mating mix.

No threonine kills the F- cells in the mating mix, * also, azide, T1 phage, and a lack of carbon source.

Hfr and Mapping

HfrH

azir (resistant to sodium azide)tonr (resistant to bacteriophage T1) lac+ (able to grow with lactose as sole source of carbon) gal+ (able to grow with galactose as sole source of carbon)

F-

azis (sensitive to sodium azide) tons (sensitive to phage T1)lac- (unable to grow on lactose) gal- (unable to grow on galactose)

Bacterial Map Distances

units = minutes

E. coli Map

• 0 minutes is at the threonine,

• 100 minutes is required to transfer complete genome,

Typical Problem

combine

combine

+

Join MapsRefer to partial maps for map distances.

11.5 minutes 26 minutes

Practice

• Insights and Solutions, #2,

• Problem 7.17, 7.18, 7.19.

Transformation

• heritable exchange brought about by the incorporation of exogenous DNA,

– usually DNA from same, or similar species.

Donor and Recipient

Not all cells are competent to receive DNA.

Competence

…a transient state or condition in which a cell can bind and internalize exogenous DNA molecules,

…often a result of severe conditions,

– heat/cold,– starvation, etc.

Competent Cell

Genes are expressed that produce proteins that, in turn, span the cell membrane.

Exogenous DNA Binds Receptor

Complementary Strand Degraded

...one strand of the exogenous DNA is degraded also.

Exogenous DNA Incorporated

Heteroduplex

Cell Divides

Transformation and Mapping

• transformed DNA is generally 10,000 - 20,000 base pairs in length,

– carries more than one gene,

• When two or more genes are received from the same transformation event, they are said to be co-transformed.

Linkage in Bacteria

• genes that are closer together, have a higher probability of being co-transformed,

– higher probability of being on same donor DNA,

– lower chance of crossover event between genes,

• probability of transformation by two separate events is low,

• linkage in bacteria refers to proximity.

Transposable Elements

…a segment of DNA that can move to, or move a copy of itself to another locus on the same or a different chromosome (hopping DNA),

…may be a single insertion sequence, or a more complex structure (transposon) consisting of two insertion sequences and one or more intervening genes.

Transposable Elementsmobile DNA

Transposon: carries one or more genes.

Why Transposons?

…the DNA sequence between the transposable elements may confer an adaptive advantage,

– or at differing dosages,

…upon mobilization, the transposon may ‘hop’ into a part of the genome that is being expressed at a higher or lower rate,

…other?

Recombinases

• Enzymes that catalyze recombination via “crossing-over” event,

– just as sister chromatids can recombine during Prophase I,

• any DNA can “cross-over” and recombine under the right circumstances.

Cre/lox Recombination

The enzyme Cre recombinase associates specifically with the loxp locus,

- the gene that codes for Cre is elsewhere in the genome, and is under transcriptional control.

Integrons

Site specific recombinase, plus adjacent recognition region

Integron Excision

Hop In, Hop Out

…the transposable elements, transposons and integrons, etc. may confer a temporary advantage,

…once the selective pressure is over, the transposable element can re-mobilize and exit a disrupted gene, and in many cases return the gene to its original state,

– may transpose to a conjugative plasmids, or near Hfr integration sites for wide spread dispersal,

…integron cassettes can also excise, and picked up by other genetic elements.

And, Self-Mutate?

…transposable elements are often mobilized during environmental stress,

– cassettes are shuttled from cell to cell, etc.

…for example: out of billions of cells, one cell may have a transposable element that inactivates a specific gene,

– upon inactivation, the cell may have an adaptive advantage.

Transpositionnormal gene, normal RNA, normal protein,

transposon inserted in gene, abnormal RNA, abnormal protein, loss of function.

T4 Bacteriophage

…infects E. coli,

Transduction

…virally mediated gene transfer from one bacterium to another,

…bacteria viruses are termed bacteriophages.

Two Bacteriophage Strategies

• Lytic,

– a type of viral life cycle resulting in the release of new phages by death and lysis of the host cell,

• Lysogenic,

– a type of viral life cycle in which the visus becomes incorporated into the host cell’s chromosome.

Lytic Cyclespecific transmembrane phage/bacteria binding sites,

virus DNA inserted into host cell,

1. host cell physiology is shut down,

2. host cell physiology is used for phage work,

3. phage DNA replicated,capsule parts made,

4. phage reassemble with repackaged DNA,

5. host cell is degraded and lyses.

Generalized Transduction

…enzymatic process which can result in the transfer of any bacterial gene between related strains of bacteria.

Phage Infects Host

Specific Binding Sites,

Phage DNA inserted,

Upon infection, host cell physiology is shut down,

we’ll follow gene C+.

Phage Hijacks the Host Cell’s Transcription/Translation

Machinery

Host cell degraded, the host chromosome is cut, Phage replicates own DNA,

makes protein head etc.,

gene C+ is present on a DNA fragment.

Cell Lyses, Phage Move On

C+ is packaged instead of phage DNA in one of thousands of new phages,

phage particle with C+ moves to another host cell.

End of the RouteHost Chromosome,

Phage DNA,

inserted in Genome,via double crossover.

packaged host DNA,

packaged host DNA,

inserted in cell,

Virulent Phages

…reproduce via the lytic cycle only.

Two Bacteriophage Strategies

• Lytic,

– a type of viral life cycle resulting in the release of new phages by death and lysis of the host cell,

• Lysogenic,

– a type of viral life cycle in which the visus becomes incorporated into the host cell’s chromosome.

Lytic vs Lysogenic

viral DNA is incorporated into the host genome.

Lysogeny

…the integration of viral DNA into the bacterial genome,

– a virus that can integrate into the genome is termed temperate,

– an integrated phage is termed a prophage.

Prophage

…non-virulent units that are inserted in the host chromosome, and multiply via binary fission along with the host DNA,

…prophage can re-enter the lytic cycle to complete the virus life cycle.

Phage Induction

…prophage express a repressor protein that inhibits further infection,

– also inhibits prophage DNA excision genes, and genes used during the lytic cycle,

…environmental cues (especially events that damage DNA) block the expression of the repressor protein,

– prophage excises and enters a lytic cycle.

Specialized Transduction

…upon excision of the prophage, adjacent host DNA is taken along,

…the completion of the lytic cycle and subsequent infection of another host moves the flanking DNA to another bacterium.

Normal Excision

Abnormal Excision

flanking DNA is removed.

Transfer to Other Cells

Bacteria are Geniuses

• Cloning: identical copies,

• Gene therapy: insertion of a healthy, or functional gene into a organism lacking a good gene,

• Harness Mutation: to deal with stress and speed evolution,

• Defense: develop genes to ward off poisons, predators, etc., then share the goods,

• Genetic engineering: inserting DNA into another organism to do your bidding (Friday),

Phage Infections

Phage Phenotypes/Genotypes

Single Phage Infection; uniform plaque morphology,

• different phage genotypes can yield different phage phenotypes.

Phage Particles Can Recombine

r+: small plaque

r-: large plaque

h+: clear plaque

h-: turbid plaque

What is a Gene?

• Bead Theory (< 1950s),

– The gene was viewed as a fundamental unit of structure, indivisible by crossing over.

– The gene was viewed as the fundamental unit of change (mutation).

– The gene was viewed as the fundamental unit of function (parts of genes were not thought to contain function).

Genetic Fine Structure

• Seymore Benzer;

– Demonstrated that a gene can be subdivided into a linear array of sites that are mutable and that can be recombined.

– Paved the way for the understanding that the smallest units of mutation and recombination are single nucleotide pairs.

rII

• A mutant T4 phage was known to produce larger, ragged plaques…

– this mutation was mapped to two genetic loci on the phage DNA molecule, rI and rII,

• rII mutants have an altered host range compared with wild-type T4.

rII Host Range

Permissive: E. coli strain B is permissive to rII-

Non-permissive: E. coli strain K() is non-permissive to rII-

rII-mutants

Infect B with two rII- mutants…

…infect K cells with resultant phage.

Control: rII- parents on K plates.

Intergenic Recombination

Co-transduce on B.

Select for wt recombinants on K.

Frequency of recombination indicates map distance.

rII6rII3rII1 rII8rII2rII5rII4rII7

X

Xx

rII7

rII8

rII1 - rII8

Why Stop There?

• Deletion Mapping: partial deletions in genes can be mapped in just the same way as other mutations…

– in fact, the site of the deletion can be determined by defining which previously mapped mutations fail to to

recombine into a wild-type gene.

Please Study, and master “A moment to Think”, pp. 269

Deletion Mapping

IntervalsA1 - A6 or B

Subintervals ...break down

interval.

Fine map with “reversible” mutations in subinterval.

What Did We Learn?

• Genes are linear arrays of sub-elements,

• the sub-units are alterable by mutation and able to recombine (average = 2.3 bases),

• mutations are not produced at all locations in a gene,

– and are found at higher frequencies at certain locations.

Coming Up Wednesday: Plant Biotechnology

…bacteria also have plasmids (T Plasmids) that they transfer to other organisms,

…upon infection, the T plasmid enters the host cell, becomes incorporated in the host genome, and the T plasmid genes become expressed,

…Agrobacterium tumefaceins transfers genes that force plants to make strange sugars, that only the Agrobacterium can digest.

Review: Friday