Specialized Transduction


Briefly describe the difference between DNA packaged in P22 generalized transducing particles and DNA packaged in lambda specialized transducing particles.

ANSWER: Important differences are:


Briefly describe the differences between a low frequency transducing (LFT) lambda dgal lysate and a high frequency transducing (HFT) lambda dgal lysate of phage lambda. [Indicate how the two types of transducing particles are obtained and the relative frequency of transducing particles in each lysate.]

ANSWER:


What are secondary attachment sites for phage lambda?

ANSWER: Secondary attachment sites are sequences where lambda can integrate if the normal attB site on the chromosome is deleted. The frequency of integration at such secondary sites is so much less than at attB that integration at the secondary attachment sites is essentially never observed if the wild-type attB site is present. See the description of lambda attachment sites under supplemental resources.


How can secondary attachment sites be used to isolate novel lambda transducing phages?

ANSWER: Abbarent excision of a lambda lysogen from a secondary attachment site adjacent to the gene of interest could yield a LFT carrying the gene. For example, to obtain lambda dthr specialized transducing phage that carry the threonine biosynthesis genes (thr), you would isolate lysogens in an E. coli del(attB) mutant, induce the excision of the prophage with UV, then use the resulting lysate at a high MOI to transduce a thr mutant strain to Thr+. This will occur at a very low frequency, but the resulting Thr+ lambda lysogens should include the desired specialized transducing phage.


There is a secondary attachment site for phage lambda within the proB gene of the E. coli proBA operon.

  1. Draw a diagram showing how a prophage integrated at this site could generate proA specialized transducing particles.

    ANSWER: See figure below.

  2. How could you use this transducing lysate to obtain a high frequency transducing (HFT) lysate that carries proA+.

    ANSWER: Using the LFT at a high MOI to isolate a double-lysogen, then induction of the double-lysogen will yield a HFT.


There is an attachment site for the lambdoid phage Phi21 adjacent to the icd gene on the E. coli chromosome. icd mutants are glutamate auxotrophs. Draw a diagram showing how a LFT containing icd specialized transducing particles could be formed following infection of a Phi21S host. Indicate the expected frequencies for any rare events.

ANSWER:

How would you test for icd specialized transducing particles? [Indicate the recipient you would use and the phenotype you would test.]

ANSWER: Use a Phi21 recA icd recipient. Select for growth on minimal medium without glutamate. [Note that the recA mutation insures that any transduction to Icd+ is due to specialized transduction, not generalized transduction.]


There is an attachment site for phage Phi80 near the supF gene on the E. coli chromosome. The supF gene encodes an amber suppressor tRNA.

  1. Draw a diagram showing how supF specialized transducing particles could be formed following infection of a 80S supF host. Note the expected frequencies for any rare events.

    ANSWER:

  2. How would you test for supF specialized transducing particles? Indicate the recipient you would use and the phenotype you would test.

    ANSWER: Note that the phenotype of the sup mutant (suppression of amber mutations) is dominant over the wild-type phenotype (no suppression). This question asks how you would identify a sup specialized transducing phage -- that is, a phage able to suppress amber mutations. Transduce a strain with auxotrophic mutations caused by amber mutations -- if the phage simultaneously suppresses two different amber mutations, the phage probably carries an amber suppressor.


Although P22 is a generalized transducing phage, it is also possible to obtain specialized transducing derivatives of P22. For example, specialized transducing particles have been obtained for the newD and supQ genes which are located adjacent to the P22 attachment site on the S. typhimurium chromosome (ataA). How could you distinguish a P22 generalized transducing lysate from a P22 specialized transducing lysate using simple genetic tests? [Be sure to give a genetic test that identifies each lysate. You can use S. typhimurium strains with any genetic markers you desire, but examining the phage in an electron microscope, DNA sequencing, or using antibodies are not acceptable answers.]

ANSWER: A distinguishing difference between generalized transduction and specialized transduction is the ability to transduce DNA from anywhere on the chromosome. Thus, you could simply test for transduction of several different genes located at different sites on the chromosome. A simple way to do this would be to test for transduction of several different auxotrophic mutations in the recipient, selecting for prototrophy.


Four different lambda dgal phage were isolated. These phage were then crossed against four lambda point mutants, selecting for wild-type recombinants. The lambda dgal phage were also crossed against four different E. coli gal mutants, selecting for Gal+ recombinants. The results of these experiments are shown in the table below (+ indicates wild-type recombinants were obtained and - indicates no wild-type recombinants were obtained).

  1. Draw the gal-bio region of an E. coli lambda lysogen, and label the bio operon, the gal operon, and the lambda prophage. Based upon the results shown in the table above, indicate the relative map positions of each of the four gal mutations.

    ANSWER: See figure below:

  2. Indicate the relative positions of each of the four lambda mutations on the lambda genome and show the region missing in each of the lambda dgal phages.

    ANSWER: See figure below:


A commonly used trick for doing complementation analysis without excess copies of the gene of interest is to clone the gene onto a lysogenic phage and integrate the phage into the chromosome. For example, the ompC gene of E. coli has been cloned onto lambda and integrated into the attB site on the chromosome. In order to study complementation between two ompC mutations, a strain is constructed with one mutant allele of the ompC gene at its normal chromosomal location and one mutant allele of the ompC gene at the lambda attachment site.

  1. You have two strains of E. coli. One strain contains an insertion mutation in the ompC gene that results in resistance to the antibiotic kanamycin (ompC::Kan). The other strain is a merodiploid with a null mutation in the chromosomal ompC gene and a lambda lysogen with a wild-type copy of the ompC gene. How could you easily move the ompC::Kan mutation into the merodiploid strain?

    ANSWER: Grow phage P1 on the E. coli ompC::Kan mutant to obtain a generalized transducing lysate, then transduce the merodiploid strain selecting for KanR.

  2. How could you determine which copy of the ompC gene acquired the insertion mutation?

    ANSWER: If excision of phage lambda yielded transducing particles that carry KanR then the mutation must be located in the lambda copy. Alternatively, if the lambda transducing particles could repair the ompC::Kan insertion in the parent strain then the mutation is not in the lambda copy.

  3. If the ompC::Kan mutation was located on the lambda lysogen, how could you move the prophage to a new strain?

    ANSWER: Induce the lambda by treating the cells with UV light, then infect cells with the lambda lysate, selecting for KanR.


Given two phage lysates -- (i) lambda gal transducing phage, and (ii) P1 phage grown on wild-type E. coli -- how could you identify each lysate using simple genetic tests? [Your answer may take advantage of E. coli strains with any genetic markers you desire, but you should be able to answer this question without proposing to examine the phage in an electron microscope, determining the DNA sequence, or using antibodies.]


A strain of E. coli is simultaneously infected with lambda gal and lambda bio transducing phages at a multiplicity of infection (moi) of approximately 5 for each phage. After the culture lyses, approximately 10% of the phage released are wild-type lambda. No wild-type lambda were found in control single infections. Explain how wild-type lambda can be generated in the double infection. Use a diagram to explain your model and indicate the cellular and/or phage factors required. Would you expect another new class of phage from this cross? If so, indicate what the structure of this phage would be.


The E. coli hfl gene affects the lysis-lysogeny decision of phage lambda.

  1. What results would you expect if you infected an E. coli hfl gal mutant with lambda dgal+ HFT? Briefly explain your answer.

    ANSWER: In the absence of the Hfl protease, a high concentration of cII would accumulate, favoring lysogeny. Thus, a high frequency of Gal+ transductants would be obtained. [See the Mcbio 316 supplement on lambda lysogeny for more discussion of the role of Hfl.]

  2. Would the MOI used affect your answer? Briefly explain.

    ANSWER: At a very low MOI, few cells would be infected so few Gal+ transductants would be obtained. However, because of the absence of the Hfl protease, the number of phage per cell will have little effect on the lysis-lysogeny decision. Note that an HFT lysate also contains wild-type phage, but these phage will also enter the lysogenic pathway.

  3. What would happen if the E. coli hfl mutant was already a lambda lysogen? Briefly explain.

    ANSWER: The cI repressor protein made by the prophage would repress the incoming phage -- no integrase would be produced so the only way the incoming phage could integrate into the chromosome would be via homologous recombination with the preexisting lysogen. Homologous recombination of the gal+ genes on the phage with the gal genes on the chromosome could yield Gal+ transductants, but the frequency of this would be low relative to the number obtained following site-specific recombination of the phage.


The thr operon is required for the biosynthesis of the amino acid threonine. Lambda specialized transducing phage were isolated which carry the thr operon. (The thr operon is not located near the normal attachment site for phage lambda on the E. coli chromosome.) Two phage lysates, a LFT lambda dthr lysate and a HFT lambda dthr lysate, were used to transduce a thr mutant to Thr+.

  1. What mutation is needed in the strain used to look for lambda insertions near the thr operon?

    ANSWER: A bacterial host strain deleted for the lambda attachment site (attB).

  2. How would you select for the Thr+ transductants?

    ANSWER: Plate the infected cells on minimal medium without threonine.

  3. What is the relative frequency of Thr+ transductants would you expect from each lysate and why?

    ANSWER: The frequency of transduction would be proportional to the frequency of Thr+ transducing particles in the lysate. The Thr+ transducing particles in the LFT lysate would probably be about 10-6 of the total phage in the population or 0.0001%. The Thr+ transducing particles in the HFT lysate would be about 50% of the total phage in the population.

  4. If the transduction was done at a low MOI, the resulting Thr+ transductants are stable. If the transduction was done at a high MOI, many of the resulting Thr+ transductants are not be stable. Why?

    ANSWER:


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    This page is maintained by Stanley Maloy, please send comments, suggestions, or questions to s-maloy@uiuc.edu
    Last modified April 26, 2000