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Lecture Outline 9/22

• Bacterial Genetics overview• Types of gene transfer:

– Conjugation– Transformation– Transduction

• Mapping genes by time of entry

Very different experimentalmodel:

• Use huge numbers of individuals(billions) To find very rare events

• Few morphological phenotypes

• Don’t count all progeny; insteaddevise ways to select and detect thefew transformants

Lederberg & Tatum (1946) Experiment demonstratingrecombination in E. coli. Recombination of 2 complimentaryauxotrophs gives rise to a strain that can synthesize all nutrients.

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F factors

• F is on a plasmid, with its own origin ofreplication

• One-way transfer, F+ to F-– Requires physical contact (conjugation)– A single strand is transferred to the recipient

cell, where it is replicated and cirularized

6-11Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

• Plasmids, such as F factors, which are transmitted viaconjugation are termed conjugative plasmids– These plasmids carry genes required for conjugation

Figure 6.3

These genes play a role in the transfer of DNAThey are thus designated tra and trb followed by a capital letter

High Frequency Recombination

High Frequency Recombination

• Hfr strains integrate F into theirchromosomes– MUCH more likely to transfer

chromosomal DNA• Complete chromosome is rarely

transferred (1/10,000)• Recipient remains F-

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Using Conjugation to mapbacterial genes

• Score frequency of exconjugants aftervarious 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

Using conjugation to mapbacterial genes:

• Begin with appropriate Hfr strains selected from F+ x F- crosses and performan interrupted mating experiment.

– 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 should you use here?

• Remove at experimental time points and agitate to separateconjugating pairs.

• Analyze recombinants with selective media.• 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.

Interruptedmatingexperiment

Variation in insertion andorientation of Hfr fragments Review

• Watch an animation from the textbookwebsite

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Generating a map for all of E.coli:

1. Location and orientationof the Hfr F+ in thecircular chromosomevaries from strain tostrain.

2. Overlap in transfer mapsfrom different strainsallow generation of acomplete chromosomalmap.

Example• Four different Hfr strains of E. coli were mated to F- recipients

to determine the time of entry of various donor markers. Theresults are shown below.– Constuct a genetic map– What is the distance between adjacent marker pairs?

Strain marker (time of entry)Hfr#1 arg (15) thy (21) met (32) thr (48)Hfr#2 mal (10) met (17) thi (22) thr (33) trp (57)Hfr#3 phe (6) his (11) bio (33) azi (48) thr (49) thi (60)Hfr#4 his (18) phe (23) arg (45) mal (55)

Fig. 14.25

Circular geneticmap of E. coli

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That is the approximatetime required for an E.coli chromosome toreplicate at 37°C

Total map units =100 minutes

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 thenew gene

Does this happen in nature?

• In E coli and Salmonella, roughly 17% oftheir genes have been acquired from otherspeices (over 100 million years . . . )

• Such “horizontal transfer” is an importantissue for the spread of antibiotic resistance

Spread of Atrizine decomposingbacteria

• Atrizine is a synthetic herbicide• A few bacteria are capable of metabolizing

Atrizine in the soil• All have nearly identical genes!