Chapter 6 - Genetic Analysis and Mapping in Bacteria Notes PDF

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Dr. Chong...

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Bacterial Genomes: ● Single chromosome ● Bacterial chromosome is usually a covalently closed circular molecule of double-stranded DNA ● Can have additional pieces of DNA organized into plasmids Types of Plasmids: ● F (fertility) plasmid - contains genes that promote their own transfer from donors to recipients ● R (resistance) plasmid - carries antibiotic resistance genes that can be transferred to recipient cells

F Plasmid

R Plasmid

Plasmid Replication: ● Many replicate independently of the bacterial chromosome so that the number of plasmids per cell can increase rapidly ● “High-copy-number” plasmids → actual number per cell is variable ● Low-copy-number plasmids are present in one or two copies per bacterial cell → usually cannot replicate independently of the bacterial chromosome 3 Processes of Genetic Transfer: 1. Conjugation 2. Transformation 3. Transduction Conjugation: ● Transfer of replicated DNA from donor to recipient ● Can pass chromosomal DNA, plasmid DNA, or both ● Donor cells may possess an F factor (F+ cells), while recipients (F- cells) lack an F factor

Conjugation

Conjugation of F+ and F- Cells

Conjugation of F+ and F- Cells: ● OriT signifies where F factor beings its transfer ● Rolling circle replication pushes T strand into recipient ● The donor cell (F+) assembles a conjugation pilus to contact the recipient cell (F-) ● The relaxosome complex binds the F factor at oriT and cleaves the T strand of the DNA ● The relaxosome partially degrades, leaving the relaxase bound at the 5’ end of the T stand. The relaxase -T strand complex binds to a coupling factor to prepare for export. Rolling circle DNA replication begins in the donor. ● The exporter moves the relaxase -T strand complex replication in the donor spools the T strand to the recipient, where it is a template for DNA replication ● Completion of replication in both cells leaves the donor (F+) unchanged and converts the recipient cell to an F+ donor state

IS Element: ● 4 Insertion sequence (IS) elements - large component of the F factor ● IS elements that are shared by an F plasmid and bacterial chromosome allow for recombination between the two ● Recombination of bacterial chromosome and F factor occurs at an IS element

Factor Integration → Hfr Chromosomes: ● Recombination of bacterial chromosome and F factor at an IS element ● F factor integration → Hfr chromosome

Recombination and Integration (rare)

Hfr Chromosome: ● F factor in Hfr strains integrates into bacterial chromosome to form the Hfr chromosome ● Formation of Hfr chromosome occurs rarely ● Integration occurs at one of multiple IS elements that are shared by F plasmids and bacterial chromosomes ● Location and orientation of F factor varies among Hfr strains

DNA Bacteria Exchange in Lab: ● Conjugation experiments involve mixing donor and recipient cells and examining exconjugants ● Exconjugants are identified by their growth on a selective growth medium ● Selective media contain compounds that permit growth of exconjugant cells of specific genotype sand prevent donor and recipient growth

Selection with Antibiotic Resistance:

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Antibiotic sensitivity and resistance is used as a tool to control the growth of bacteria If recipient cells contain an R plasmid conferring resistance to streptomycin (str^R), donor cells that lack the plasmid will be sensitive to streptomycin (str^R) Plating on media containing streptomycin will select against donor cells, but not recipients or exconjugants Bacteria carrying the plasmid survive in the dish with streptomycin → when all other cells die, then they know that all living cells now have plasmid

Prokaryotic Mapping: ● No meiosis ● Possible to use conjugation to do genetic mapping

Interrupted Mating and Time-of-Entry Mapping: ● Interrupted mating - end of conjugation by breaking the conjugation pilus ● Stops mating before the Hfr chromosome can be completely transferred to the recipient cell ● Experiments that test for gene transfer at timed intervals (time-of-entry mapping) are used to determine the distances between genes ● Genes closer to the oriT (accounting for direction) enter the recipient cell sooner

Time-of-Entry Mapping: ● An allele has been transferred because scientists stop the mating ● Place the cells on selective media ● If the cell received required allele, it will grow ● DNA from donor cell will recombine into the recipient genome forming an exconjugant cell

Time-of-Entry Mapping Outcomes of Hfr x F- Mating:

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Transfer one or more donor alleles into recipient chromosome occurs by homologous recombination Forms an exconjugant chromosome Allows for time-of-entry mapping F factor is not fully transferred during the mating

Consolidation of Hfr Maps: ● Mapping information for a single Hfr strain is limited ● Each Hfr strain can transfer genes in one direction only ● Because mating is interrupted, the likelihood of gene transfer drops off quickly with distance from oriT ● Multiple Hfr strains must be used in interrupted mating experiments to map all of the genes in a species

Direction of Gene Transfer

Overlapping Linear Maps: ● Data from each Hfr strain is used to create partial overlapping maps ● Each Hfr strain is possible because of IS element recombination Circular Maps: ● If Hfr4 wasn’t available, there would be no circular map ● The consolidated map shows gene order and the cumulative number of minutes, site, and orientation of each integrated F factor

Overlapping Linear Maps

Circular Map

Creation of F’ Strains: ● F’ donor - contains a functional but altered F factor derived from imperfect excision of the F factor from an Hfr chromosome ● F’ factor - contains all of its DNA plus a segment of the bacterial chromosome ● F’ cells - donor cells carrying F’ factor ● F’ factor created by aberrant excision

Creation of F’ Strains

F’ x F- Mating: ● Partial Diploid → F’ exconjugant has 2 copies of the lac gene, but one copy of most other genes ● The exconjugant is lac+/lac- partial diploid and has acquired the ability to grow on a lactose medium → since F’ plasmid transfer was complete, the exconjugant can act as an F’ donor

F’ x F- Mating

Transformation: ● Transformation - uptake of DNA from the environment ● Transformation occurs when a recipient cell takes up fragments of donor DNA from the surrounding growth medium

Transformation

Transformation of a Competent Bacterium (a-) by Donor DNA (a+): 1. Donor DNA binds at the receptor site - one strand is degraded as it enters the recipient cell 2. Transforming strand pairs with the homologous region of the recipient chromosome 3. Transforming strand displaces a recipient strand, forming complementary heteroduplex DNA (a-/a+) - the excess strand degrades 4. DNA replication and cell division produce one transformation and one non transformant

GMOs: ● Arctic apple → down-regulates a gene (polyphenol oxidase) that results in no-browning ● Bt corn → produce bacterial protein that kills insects who eat plant tissue ● Rainbow papaya → viral resistance against Papaya ringspot virus

Transduction: ● Transduction - transfer of DNA from one bacterium to another by a viral vector 1. Bacteriophage attaches to the donor cell 2. Phage DNA injection leads to donor chromosome fragmentation 3. Transducing phages package donor DNA instead of phage DNA 4. Transducing phage attaches to a recipient cell 5. Donor DNA injected and recombined with the recipient chromosome 6. Transductant cell produced by recombination

Transduction

Bacteriophage Life Cycles: ● Tiny viral particles that infect bacteria ● Composed of an icosahedral head, hollow protein sheath, and sometimes a set of tail fibers ● Head contains a single chromosome ● Replication and gene expression require enzymes and factors of the host cell

T4 and Gamma Bacteriophage

Lytic Life Cycle of Temperate Bacteriophage: 1. Phage attaches to host cell 2. Phage injects DNA through hollow tail 3. Replication of phage chromosome occurs → host DNA breaks down 4. Under the direction of phage genes, transcription and translation produce new phage components 5. DNA and proteins are assembled into progeny phages 6. Progeny phage particles are released by lysis from host bacteria ● Bacteriophages use host enzymes and ribosomes to replicate, transcribe, and translate Lysogenic Cycle: ● Some bacteriophages (temperate phages) have an alternate, temporary life cycle involving integration of the phage chromosome into the bacterial chromosome ● Lysogeny - integration ● Lysogeny can persist for many bacterial cell cycles, but eventually comes to an end, triggering the lytic cycle 1. Phage attaches to host cell 2. Phage injects DNA through hollow tail 3. Integration of phage DNA into host chromosome 4. Multiple divisions and generations occur → prophage DNA is copied when cell divides 5. Excision of prophage from host chromosome 6. Lytic cycle resumes

Bacteriophage Life Cycle → Lytic and Lysogenic

Generalized Transduction: ● Generalized transducing phages → package random piece of donor bacterial DNA into progeny phage heads ● Error occurs because DNA to be packaged is selected only based on size ● Phage P1 is a well-student phage that infects E. coli ● Phage P1 is a producer of generalized transducing phages Example of Transduction by P1 Phage: 1. P1 phage infects a met+, his+ donor cell 2. Phage chromosome is replicated and phage proteins are expressed → donor chromosome fragments 3. Progeny phage produces normal phages with the phage chromosome and transducing phages with a fragment of the donor chromosome 4. Lysis releases normal and transducing progeny phages 5. A met+ transducing phage infects a met- recipient cell and injects the donor DNA fragment 6. Homologous recombination at 2 crossover points exchanges segments between the donor fragment and the recipient chromosome 7. The transductant is met+, his- → excised DNA with met- is degraded ● Thus, transduction can change the genotype of the recipient cell

Cotransduction Mapping: ● The close 2 genes are on the donor chromosome, the more likely they will be transduced to a recipient together (cotransduction) ● Cotransduction frequency depends on the distance between 2 genes ● To find cotransductants, researchers may have to examine many colonies ● Host chromosome is broken apart → genes close together end up on the same fragment

Lateral Gene Transfer and Genome Evolution: ● Lateral gene transfer (LGT) → transfer of genetic material between individual bacteria or archaea and other organisms ● Studies of LGT find that more than 12% of genes in a genome are a result of LGT, on average, with a wide range ● Genes coding for cell surface proteins, DNA-binding proteins, and proteins related to pathogenicity are more likely to undergo LGT

LGT Between Domains: ● LGT occur among bacteria and also between bacteria and eukaryotes ● Evolutionarily, an example of LGT involves the endosymbiosis that led to mitochondria and chloroplasts in eukaryotes ● Bacteria have inserted genes into sweet potatoes → sweet potatoes then transcribe those genes LGT in Genomes: ● LGT is identified by presence of DNA-sequence features in regions of a genome that are uniquely different from the rest of the genome ● Genomic islands - unique regions of genome 2 Common Characteristics of Lateral Gene Transfer Regions: 1. Island region has a ratio of G-C base pairs to A-T base pairs that is substantially higher or lower than the average in the rest of the genome 2. Group of genes in the island are more similar to genes of distantly related species than a closely related species...