GEB 204

General Genetics and Genetic Analysis

Microbial Genetics
Lecture 12

Arindita Das
Lecturer
Department of Genetic Engineering and
Biotechnology
East West University
 Generalized transduction
• In 1965, K. Ikeda and J. Tomizawa found that when a donor cell is lysed by E. coli phage P1, the
bacterial chromosome is broken up into small pieces.
• Occasionally, the newly forming phage particles mistakenly incorporate a piece of the bacterial
DNA into a phage head in place of phage DNA. This event is the origin of the transducing phage.
• A phage carrying bacterial DNA can infect another cell. That bacterial DNA can then be
incorporated into the recipient cell’s chromosome by recombination (Figure 5-27).
• Because genes on any of the cut-up parts of the host genome can be transduced, this type of
transduction is by necessity of the generalized type.
• Phages P1 and P22 both belong to a phage group that shows generalized transduction. P22 DNA
inserts into the host chromosome, whereas P1 DNA remains free, like a large plasmid. However,
both transduce by faulty head stuffing.
• Generalized transduction can be used to obtain bacterial linkage information when genes are close
enough that the phage can pick them up and transduce them in a single piece of DNA.
• The greater the cotransduction frequency, the closer two genetic markers must be.
The mechanism of generalized transduction
 BEHAVIOR OF THE PROPHAGE

• Phage  has unusual effects when cells lysogenic for it are used in crosses.
• In the cross of a nonlysogenic Hfr with a lysogenic F− recipient [Hfr  F− ()], lysogenic F−
exconjugants with Hfr genes are readily recovered.
• However, in the reciprocal cross Hfr()  F− , the early genes from the Hfr chromosome are
recovered among the exconjugants, but recombinants for late markers are not recovered.
Furthermore, lysogenic exconjugants are almost never recovered from this reciprocal cross.
• What is the explanation?
• The observations make sense if the  prophage is behaving like a bacterial gene locus (that
is, like part of the bacterial chromosome). Thus, the  prophage would enter the F− cell at a
specific time corresponding to its position in the chromosome.
• Earlier genes are recovered because they enter before the prophage. Later genes are not
recovered, because lysis destroys the recipient cell. In interrupted-mating experiments, the 
prophage does in fact always enter the F− cell at a specific time, closely linked to the gal
locus.
• In an Hfr()  F− cross, the entry of the 
prophage into the cell immediately triggers the
prophage into a lytic cycle; this process is called
zygotic induction (Figure 5-29).
• However, in the cross of two lysogenic cells
Hfr()  F− , there is no zygotic induction.
• The presence of any prophage prevent another
infecting virus from causing lysis. The prophage
produces a cytoplasmic factor that represses the
multiplication of the virus.
  INSERTION
The interrupted-mating experiments described above showed that the  prophage is part of
the lysogenic bacterium’s chromosome. How is the  prophage inserted into the bacterial
genome?
In 1962, Allan Campbell proposed that it inserts by a single crossover between a circular
chromosome and the circular E. coli chromosome, as shown in Figure 5-30.
The crossover point would be between a specific site in , the  attachment site, and an
attachment site in the bacterial chromosome located between the genes gal and bio, because
 integrates at that position in the E. coli chromosome.
The crossing-over is mediated by a phage encoded recombination system.
One attraction of Campbell’s proposal is that from it follow predictions that geneticists can
test. For example, integration of the prophage into the E. coli chromosome should increase
the genetic distance between flanking bacterial markers, as can be seen in Figure 5-30 for
gal and bio.
In fact, studies show that lysogeny does increase time-of-entry or recombination distances
between the bacterial genes. This unique location of  accounts for its specialized
transduction.
Model for the integration of phage  into the E. coli
chromosome. Reciprocal recombination takes place
between a specific attachment site on the circular  DNA
and a specific region on the bacterial chromosome
between the gal and bio genes.
 Mechanism of specialized transduction
• As a prophage,  always inserts between the gal region and the bio region of the host chromosome
and in transduction experiments, as expected,  can transduce only the gal and bio genes. How does
carry away neighboring genes?
• The explanation lies again in an imperfect reversal of the Campbell insertion mechanism, as for
generalized transduction.
• The recombination event between specific regions of  and the bacterial chromosome is catalyzed
by a specialized enzyme system. The  attachment site and the enzyme that uses this site as a
substrate dictate that  integrates only at that point in the chromosome (Figure 5-31a).
• Furthermore, during lysis the  prophage normally excises at precisely the correct point to produce
a normal circular  chromosome as seen in Figure 5-31b(i).
• Very rarely, excision is abnormal owing to faulty outlooping and can result in phage particles that
now carry a nearby gene and leave behind some phage genes [see Figure 5-31b(ii)]. The resulting
phage genome is defective because of the genes left behind, but it has also gained a bacterial gene
gal or bio. These phages are referred to as  dgal ( -defective gal) or  dbio.
• The abnormal DNA carrying nearby genes can be packaged into phage heads and can infect other
bacteria. In the presence of a second, normal phage particle in a double infection, the  dgal can
integrate into the chromosome at the  attachment site (Figure 5-31c). In this manner, the gal genes
in this case are transduced into the second host.
Specialized transduction
mechanism in phage 
(a) A crossover at the specialized
attachment site produces a lysogenic
bacterium. (b) Lysogenic bacterium
can produce a (i) normal  or, rarely,
(ii) dgal, a transducing particle
containing the gal gene. (c) gal+
transductants can be produced by
either (i) coincorporation of dgal
and  (acting as a helper) or, rarely,
(ii) crossovers flanking the gal gene.
Do bacterial cells ever pair up for any type of sexual cycle?
Yes, there is a mating-like process called conjugation, governed by the F plasmid. All
mattings are between one cell with F and one cell without F.
Do bacterial genomes ever show recombination?
Yes, recombinants can be detected. They are relatively rare but can be demonstrated using
selection procedures: for example, selection on plates for prototrophic recombinants
between two auxotrophic parents.
If so, in what ways do genomes become associated to permit recombination?
Conjugation, transformation, and transduction are all ways of bringing together genes from
different bacterial parents.
Can bacterial and viral chromosomes be mapped using recombination?
Yes, all bacterial merozygotes can be used for recombinant frequency-based mapping.
There are also some unusual methods, for example, mapping by time of entry during
conjugation, and frequency of co-transduction by phage. Virus chromosomes can be
mapped, too.
Do the genomes of bacterial viruses ever show recombination?
Yes, if a bacterial host is infected simultaneously by two phage types, recombinant
phage are found in the lysate.

Do bacterial and viral genomes interact physically in any way?

Yes, temperate phages can pick up bacterial genome fragments during lysis and
transfer them to other bacteria when they reinfect. Some phages pick up only one
specific region; others can pick up any segment that can be stuffed into a phage
head.