Intro to Bacterial genetics and mapping

Comparison of Bacterial genetics with the Eukaryotic genetics we have done so far:

• Very different experimental model:
• Use huge numbers of individuals (billions) To find very rare events
• Few morphological phenotypes
• Don't count all progeny; instead devise ways to select and detect the few transformants

Three mechanisms of genetic exchange:

• Conjugation
• Transformation
• Transduction

Conjugation

• Requires physical contact
• Mediated by F factors
• F is on a plasmid, with its own origin of replication
• One-way transfer, F+ to F-
• A single strand is transferred to the recipient cell, where it is replicated and circularized

F' factors are F+ plasmids with a bit of bacterial DNA

Hfr strains integrate F into their chromosomes

Why does that matter?

Using Conjugation to map bacterial genes

• Score frequency of transconjugants after various times
• Time will be the unit of genetic distance
• E. coli has a genome of about 100 min
• How do you determine time of entry?
• • Plot recombination vs minutes and extrapolate back to the origin
• See fig ??

Example:

HfrH thr+ leu+ aziR tonR lac+ gal+ strR

F- thr leu aziS tonS lac gal strS

Mix 2 cell types in medium at 37°C.

• What kind of selection (if any) should you use here?
• Remove at experimental time points and agitate to separate conjugating pairs.
• Analyze recombinants with selective media.
• What selection should you use in this step?
• Order in which genes are transferred reflects linear sequence on chromosomes and time in media
• Frequency of recombinants declines as donor gene enters recipient later.

Hfr strains:

• Different Hfr strains are characterized by variation in insertion and orientation of Hfr fragments
• Complete chromosome is rarely transferred (1/10,000)
• Recipient remains F-

Example

Four different Hfr strains of E. coli were mated to F- recipients to determine the time of entry of various donor markers. The results are shown below.

• Constuct a genetic map
• What is the distance between adjacent marker pairs?
• • Hfr#1 arg (15 min) thy (21 min) met (32 min) thr (48 min)
  • Hfr#2 mal (10 min) met (17 min) thi (22 min) thr (33 min) trp (57 min)
  • Hfr#3 phe (6 min) his (11 min) bio (33 min) azi (48 min) thr (49 min) thi (60 min)
  • Hfr#4 his (18 min) phe (23 min) arg (45 min) mal (55 min)

We started to set this problem up in class-I'll leave it to you to finish it.

Transformation

(Remember the Griffith's experiment?)

• Bacteria can pick up free DNA
• Cells must be "competent" to be transformed
• DNA enters the cell as single-stranded DNA
• Recombines with the homologous bacterial sequence to form a heteroduplex
• • How does that differ from a diploid?
• After replication, one descendant carries the new gene

Does this happen in nature?

• In E coli and Salmonella, roughly 17% of their genes have been acquired from other speices (over 100 million years . . . )
• Such "horizontal transfer" is an important issue for the spread of antibiotic resistance
• We briefly discussed an example of the transfer of the ability to catabolize atrizine- various bacteria species around the world have nearly identical genes.

Transduction via phage

Bacteriophage (e.g. Lambda, T1, T2, T4, etc) are viruses of bacteria

• Some are "virulent" (or lytic), others are "temperate (or lysogenic)
• Temperate phage have a prophage stage where they are integrated into the bacterial genome
• Transfer of genes between bacteria via phage is quite rare, but because there are so many cells you can find transductants.
• As an exercise, you might list all of the rare events that are required

Generalized transduction

• Random Piece of bacterial DNA incorporated into the phage
• Only small segments of chromosome can be taken up (<2 min)
• To map genes via transduction, look for co-transduction. If two genes commonly co-transduce, then they must be close together.

To be continued next time . . .