Chapter 6: Genetic Analysis and Mapping in Bacteria and Bacteriophages

1. Know the difference in each cell type Hfr vs F+ vs F- vs F’ i. Which of these can be a donor? ii. Which cannot be a donor cell? iii. Where is the plasmid or DNA for each of these cell types? Is it integrated or a true plasmid? Can it reform the plasmid?

Vertical gene transfer

(eukaryotes) parents → offspring

• Mitosis and meiosis events

Horizontal gene transfer

(prokaryotes) bacteria and 3 methods

autotrophic

can make own C source

Heterotrophic

needs to take in a C source

Auxotrophic

requires supplement

Prototrophic

WT Bacteria; synthesize all essential nutrients

Plasmids

• carried by bacteria

• small double-stranded circular DNA molecules containing nonessential genes

• replicate

• easily modified

• used in a variety of recombinant DNA replications (delivers DNA)

• help overcome stres

• indispensable

F (fertility) plasmid

• contains genes that promote their own transfer from donors to recipients

R (resistence) plasmid

• carries antibiotic resistance genes that can be transferred to recipient cells

Conjugation

• one-way transfer of genetic material from a donor bacterial cell to a recipient cell

  • DNA transferred may be a plasmid, a portion of the bacterial chromosome DNA or both

• requires sex plus and cell to cell contact

• initiated only by donor cells

• DNA transferred through conjugation bridge

Conjugation pilus

forms between the donor and recipient cells

Relaxosome

• binds the origin of transfer (oriT) on the F factor and cleaves one phosphodiester bond on the T (transfer) strand of the F factor

• partially degenerates, leaving relaxase attached to the free 5’ end of the T strand (nicked strand)

• responsible for cutting to move over the F factor

Relaxase

• facilitates movement of the T strand through the conjugation plis and into the recipient cell

Rolling circle replication

• starts at oriT

• accompanies T stand transfer across the pilus

• uses non transferred DNA strand a s a template

• displaces the 5’ end of the T strand, freeing it

• ends with both cell containing a complete double-stranded F factor

Donors

F⁺ cells

Recipients

F^- cells

F Factor conjugation

only the F factor is transferred to the F^- strain

• F⁺ transfers plasmid

• F^’ transfers plasmid plus bacterial genes

Hfr conjugation

chromosomal DNa is transferred first, the F factor last

Conversion

the transfer of an F factor to a F^- strain, which converts the strain to F⁺

Recombination

the transfer of bacterial genes from an Hfr or F^’ strain to an F^- strain

F (fertility) factors

are cytoplasmic episomes needed of conjugation

F^’ factors

are F factors that contain some genes from the bacterial chromosome

• donor

• Transfers F’ plasmid and extra bacterial genes

• creates partial diploid cells

prototroph

Prototrophs are strains of organisms that do not require a given compound for growth: they can survive on minimal media

auxotroph

• require media supplemented with specific substances not required by the wild-type organisms

F⁺

An F⁺ cell that has a free fertility (F) factor

• has F plasmids in the cytoplasms freely without integrating into bacterial chromosomes

• Donor

• exists as an independent plasmid

• not integrated into chromosome

Hfr cell

has the fertility factor integrated into its bacterial genome

• can transfer genes in one direction only (donor)

• multiple Hfr strains must be used in interrupted mating experiments to map all of the genes in a species

• part of chromosome transfers, F factor begins transferring first

recipient remains F^-

Outcomes of Hfr x F^- Mating

• transfer of one or more donor alleles into the recipient chromosomes occurs by homologous recombination

• forms an exoconjugant chromosome

• F fafot is not fully transferred during the mating

• recipient cell is not converted into a donor cell

Interrupted mating

• the cessation of conjugation by breaking the conjugation pilus

• - stops mating before the Hfr chromosome can be completely transferred to the recipient cell

time-of-entry mapping

are used to determine the distances between genes

• each gene will transfer in a specific order

• genes closest to oriT will transfer earlier and be incorporated more frequently into recipients

Order of the remaining genes

• was determined by their time of first appearance in the exconjugant cells

F Factor Integration

• can take place at any IS element on a bacterial chromosome

• F factor can be oriented in either of two directions

Steps of Her Map Consolidation

• Partial time-of-entry maps from multiple Hfr stains are examined

• maps from each strain are consolidated by identifying overlap regions from each strain

• realist is a circular map of the donor chromosome

Transformation

occurs when a recipient cell takes up a fragment of donor DNA from the surrounding environment

• no sex pilus needed, no cell contact, or virus involved

• it’s taken up through cell membrane

• used to produce accurate maps of bacterial genes

• used in laboratories to introduce DNA into microbial cells, plants, and animal cells

• incoming dNA aligns with homologous sequence, integrated by homologous recombination

Steps in Transformation

1. The passage of dsDNA into a recipient is accompanied by degradation of one of the strands

2. The remaining strand “donor pieces” aligns with complementary regions of the recipient chromosome

3. The alignment of donor and recipient DNA triggers excision of one strand of recipient DNA and replacement with donor DNA, forming a heteroduplux

4. After subsequent DNA replication and the cell division cycle, one

   daughter cell is a transformed cell

Transformant

one daughter cell is a transformed cell

Mapping by Transformation

• free DNA is picked up by competent bacterial cell and used for mapping

  • need at least 2 genes and 2 strains

  • mix DNa from one strain with second strain

  • select only transformants

  • genes close to each other will be transformed frequently

Co-transformation frequency

# double transformants / total transformants

• the simultaneous transformation of two or more genes

Transduction

the transfer of genetic material from a donor to a recipient cell by way of a bacteriophage

• virus carries bacterial DNA from one cell to another

• Any gene may transger

• Specific genes near prophage insertion site transfer, only nearby genes transfer

• Transferred DNa recombines with chromosome, require homologous sequences

Transductant

formed by integration of donor DNA into the recipient cell’s chromosome by homologous recombination

Bacteriophage

tiny viral particles that infect bacterial host cells

• composed of an icosahedral head, hollow protein sheath, and sometimes a set of tail fibers

• cannot replicate

  • no energy metabolism, need to use a host for the protein and other compounds to reproduce the DNA

lytic cycle (virulent cycle)

leads to lysis of the host cell and release of progeny phage

lytic cycle step process

1. Attachment of the phage to the host cell

2. Injection of the phage chromosome into the host, followed by circularization of the phage chromosome

3. Replication of phage DNA using host enzymes and other proteins

4. Transcription and translation of phage genes, and subsequent production of heads, sheaths, and tail fibers for assembly of progeny phage

5. Packaging of phage chromosomes into phage heads

6. Lysis of the host cell and release of progeny phage particles

temperate phages

• has an alternates temporary life cycle involving integration off the phage chromosome into the bacterial chromosomes

lysogen

can persist for many bacterial cell cycles, but eventually comes to an end, and the lytic cycle is triggered

Steps of the lysogenic cycle

1. attachment of the phage particle to the host cell

2. Injection of the phage DNA into the host, followed by phage-chromosome circularization

3. Integration of the phage chromosome into the host chromosome at a specific DNA sequence found in both chromosomes (no replication of phage DNA)

4. Excision of the prophage in response to an environmental signal through a reversal of the site-specific integration

5. resumption the lytic cycle, beginning with phage-chromosome replication

Generalized transducing phages

package a random piece of donor bacterial DNA into progeny phage heads

• this error is because the packaging mechanism does not discriminate based on sequence, instead chooses by length

Steps of Generalized Transduction

1. A normal P1 phage attaches to a donor bacterium and injects its DNA into the cell

2. Replication of the phage chromosome is followed by transcription and translation to produce phage proteins

3. Progeny phages are assembled normally, but some phages (generalized traducing phage) receive a host DNA fragment instead of phage DNA

4. Host cell lysis releases all the progeny phages

5. Generalized transducing phages (incorrectly packaged) attach to new recipient cells and inject their DNA

6. In each recipient, homologous recombination occurs between the donor fragment and the recipient chromosome

7. A stable transductant strain results

Cotransduction

• the closer two genes are on the donor chromosome, the more likely they will be transducer to a recipient together

• Each allele can be individually transduced, but sometimes simultaneously alleles go together to be ….

• researchers examine many colonies to find them

Cotransduction frequency

depends on the distance between two genes

mutants complement

• on different genes

no complementation

• no plaques

• mutants on same gene

• no growth

homeoallelic

2 or more alleles in different organisms have a mutation in the same place on gene (same base pairs)

• done by examining the rates of rare crossovers

intragennic recombiantion

• produces wild-type and double-mutant progeny phages

Deletion Mapping

done by removal of segments of the gene to find location of a mutation

revertible mutations

caused by point mutations and could undergo spontaneous mutation back to wild-type

nonrevertible mutants

partial deletion mutants

F^- Cell

Does not contain the F plasmid

cannot initiate conjugation

• can be a recipient, not a donor

F⁺ + F^- →

F⁺ + F⁺

Griffith Experiment

Studied Streptococcus pneumoniae

• Smooth and Virulent → S strain

• Rough and Nonvirulent → R strain

Heat killed S + living R

Living S cells appeared

Lederberg and Tatum’s Experiment

worked with auxotrophic strains of E. coli

1. grew both strains together

2. mixed them together

3. allows time for interaction

4. plated cells on minimal medium

Results:

• New bacteria must have received genes from another cell

• genetic recombination occurs in bacteria

• Bacteria exchange DNA

Davis U-tube

Physical contact between bacterial cells is necessary

Conjugation requires cell to cell contact

Avery, MacLeod, McCarty

DNa is the transforming principle

Benzer

Recombination can occur within genes