This document discusses various mechanisms of horizontal gene transfer in bacteria, including conjugation, transformation, and transduction. It provides details on the genes and proteins involved in these processes, such as the tra genes for conjugation and the com genes for competence in transformation. Examples are given for using these mechanisms to map genes and determine genetic distances. Order of events in phage lifecycles and the difference between lytic and lysogenic cycles are also covered.

1. Genetic Recombination in
Bacteria and Gene mapping
By
Mr. K.C.More
Assistant Professor
Department of Botany
SGB Amravati University, Amravati (MS)
India.

2. Horizontal vs. Vertical gene transfer

3. Overview mechanisms

9. Transconjugants= recipient cell with received DNA
Promiscuos plasmids= ability to transfer DNA between
unrelated species

10. 25 tra genes
Determines
expression of sex
pilli, synthesis, and
transfer of DNA .
Tra genes are two
types:
Mating pair formation
(MPF) also include
Type IV secretion
system by which DNA
and proteins pass
and
Dtr (involved in DNA
transfer)

11. 5 to 3 prime direction
Relaxase makes cut on
origin of transfer in
plamid

22. When Recipient is partialy diploid such
transfer= Sexduction

28. Testing for Nutritional Requirements
Replica plating transfers the pattern ofReplica plating transfers the pattern of
bacterial colonies to test plates.bacterial colonies to test plates.

33. Problem no.1).
In E. coli, four Hfr strains donate the following genetic markers shown in the order
donated:
All of these Hfr strains are derived from the same F+
strain.
What is the order of these markers on the circular
chromosome of the original F+
?
Problems on Interrupted mating

34. The two-step approach that works for solution
(1)deter-mine the underlying principle, and
(2) draw a diagram. Here the principle is clearly that each Hfr strain donates
genetic markers from a fixed point on the circular chromosome and that the earliest
markers are donated with the highest frequency.
Because not all markers are donated by each Hfr, only the early markers must be
donated for each Hfr. Each strain allows us to draw the following circles:

35. From this information, we can consolidate each circle
into one circular linkage map of the order
Q, W, D, M, T, P, X, A, C, N, B, Q.

36. Problem no. 2.
In an Hfr × F −
cross, leu+
enters as the first
marker, but the order of the other markers is
unknown. If the Hfr is wild type and the F−
is
auxotrophic for each marker in question, what is
the order of the markers in a cross where leu+
recombinants are selected if 27 percent are ile+
,
13 percent are mal+
, 82 percent are thr+
, and 1
percent are trp+
?

37. We have selected for the earliest marker in this cross, the
frequency of recombinants is a function of the order of entry
for each marker. Therefore, we can immediately determine
the order of the genetic markers simply by looking at the
percentage of recombinants for any marker among the leu+
recombinants. Because the inheritance of thr+
is the highest,
this must be the first marker to enter after leu.
The complete order is leu, thr, ile, mal, trp.

38. Two bacterial strains were obtained with the following genotypes:
Hfr: ala- leu+aziSStrS
F-: ala+ leu-aziRStrR
After an uninterrupted conjugation, you want to select F- recombinants that are ala+ leu+.
Which of the following media would you use for this selection?
A) Complete medium containing streptomycin
* B) Minimal medium containing streptomycin
C) Minimal medium containing leucine and streptomycin
D) Minimal medium containing sodium azide and leucine
E) Minimal medium containing alanine, leucine, and streptomycin
If a met-thr- Hfr strain is mated with an F- of genotype leu- thi-, the prototrophic
recombinants can be detected by plating the mixture on
A) leucine and methionine.
B) threonine and thiamine.
C) leucine and thiamine.
D) methionine, threonine, leucine, and thiamine.
E) minimal medium.

39. 10.From one F+ strain the following three Hfr strains were derived, each shown with the first
three markers transferred during an Hfr × F- cross:
Hfr 1 . . . D A F →
Hfr 2 . . . E B F →
Hfr 3 . . . E C D →
The order of the genes on the bacterial chromosomal circle must be which of the
following? (A is shown at both ends to represent circularity. Assume that the Hfr picks up
all intermediates between any two represented genes.)
* A) A D C E B F A
B) A B C D F E A
C) A C D F E B A
D) A E F B C D A
E) A F B D E C A

40. An Hfr strain of E. coli with the genotype gly+ aziR StrS is mated with an
F- strain of E. coli of genotype gly- azi S StrR. Gly refers to the amino acid
glycine, azi refers to sodium azide, and Str refers to the antibiotic
streptomycin, where S is sensitive and R is resistant. Conjugation occurs
and the progeny are screened on a selective medium to detect
recombinants. If you wanted to select for the F- recombinant genotype
gly+aziR StrR, you should use a minimal medium containing
A) streptomycin.
B) sodium azide.
C) glycine.
D) glycine and streptomycin.
E) streptomycin and sodium azide.
F) Glycine, sodium azide, and streptomycin.

41. Transformation
DNA taken up from external environment
Transformation

42. Discovery

44. •The protein involved in transformation of these Gram +ve bacteria is a
product of com
•In Bacillus subtilis , the com gene are organized into several operons.
•The product of com A and com K are involved in regulation of
competence and other com E, com F and comG encodes structural
protein for uptake of DNA.
•The first gene of com E operon, com EA encodes the protein that
directly binds extracellular double stranded DNA.
•The com F gene encodes the protein that translocates DNA into cell.
for example; Com FA is an ATPase that translocate DNA into cell.
•The com G gene of comG operon encodes protein that
form pseudopilus that helps to move DNA through channel.
•The com E, com F and com G operon are under transcriptional
control of com K operon.
•Com K is a transcriptional factor that is regulated by com A
•Some other genes involved in transformation are nuc A gene that
encodes nuclease enzyme which cuts extracellular dsDNA to single
stranded,
•Single strand binding protein and Rec A gene which helps in
recombination of transforming DNA with chromosome of recipient
bacteria.

46. Transformation

47. Genetic Analysis Using Transformation
Determining genetic distance with
transformation mapping
Transform bacteria with DNA containing two markers
(e.g. his-
, met-
) in addition to penicillin sensitivity.
Select transformants on minimal medium + penicillin
to kill non-transformants.
Plate survivors on complete medium to test for his-
,
met-
.

48. Genetic Analysis Using Transformation
Determining genetic distance with
transformation mapping
Problems in Gene Transfer #4
DNA is isolated from E. coli strain A (his-
met-
pens
) and used
to transform strain B (his+
met+
pens
). Transformants are
selected on minimal medium + penicillin to kill his+
met+
cells and survivors are plated on complete medium. The
classes and numbers of cells obtained are listed below.
Determine the recombination frequency between the his
and met genes.

49. Genetic Analysis Using Transformation
Determining genetic distance with
transformation mapping
Problem #4
Rf = number of single transformants
total number of transformants

51. Transduction

55. Componenets of Bacteriophage

57. Life cycle of Bacteriophage

69. Complete vs abortive transduction

73. ?

75. Gene Transfer Processes for Bacteria and Their Viruses
Three-factor transduction
Problems in Gene Transfer #6
Transducing phages that infected an A+
B+
C+
cell are
used to infect an A-
B-
C-
cell. Transductants receiving
the A+
marker were tested for the presence of B+
and C+
. The classes and numbers of transductants
observed is shown below. Determine the gene
order and the cotransduction frequencies for A+
with B+
and A+
with C+
.

76. Gene Transfer Processes for Bacteria and Their Viruses
Three-factor transduction
Problem
AA++
BB++
CC++
4545
AA++
BB++
CC--
8080
AA++
BB--
CC++
11
AA++
BB--
CC--
300300
Select for ASelect for A++
Test for BTest for B++
andand CC++

77. The correct order of events in growth of a lytic phage, after DNA
injection, is:
a. DNA replication, Transcription of phage genes, Lysis.
b. Repressor binding to operators, Inhibition of transcription, Integration.
c. Transcription ofphage genes, Translation of phage proteins, Assembly.
d. Circularization of phage chromosome, Translation of integrase enzyme,
Lysis

78. A temperate phage chromosome which has been integrated into the
host chromosome
a.is a prophage
b.is DNA
c.maintains the potential to become a phage
d.all the above
Transduction in bacteria
a.requires the isolation of chromosome fragments
b.can be catalyzed by the fertility factor
c.requires the use of phages to move DNA from donor to recipient
d.all the above

79. Specialized transduction can be distinguished from generalized
transduction by the fact that
a.homologous recombination is involved in the former but usually not the latter
b.transducing particles are involved in the former but usually not the latter
c.a selective medium is required to demonstrate the former but usually not the
latter
d.lysogeny is involved in the former but usually not the latter
In bacteria, plasmids can be transferred from one cell to another by
a.conjugation
b.restriction
c.deletion
d.all the above

80. Which type(s) of genetic transfer lead to incorporation of new
DNA into the bacterial chromosome?
I. Conjugation mediated by F factor
II. Hfr mediated conjugation
III. Transduction
IV. Transformation
A) I and II only
B) III and IV only
C) II, III, and IV
D) I, II, III, and IV
E) None of these.

81. How would you expect a mutation in a gene encoding a
tail fiber protein would affect the plaque phenotype of T4?
A) It could lead to very large plaques.
B) It would be unlikely to have an affect on plaque
formation.
C) It could lead to smaller than normal plaques.
D) A and B.
E) None of the above.

82. How is genetic transfer different from sexual
reproduction?
A) In genetic transfer, genetic material is transferred from one
individual to another.
B) In sexual reproduction, genetic material from two individuals is
mixed equally.
C) In both processes, the individual donating genetic material retains
their original complement of genetic material.
D) None of these.

83. A researcher would like to map the location of galE and trpA genes in a
new species of bacterium that appears to be closely related to E. coli. He
decides to use cotransduction, and generates appropriate donor and
recipient strains. He is disappointed when cotransduction is not seen in
his experiement. What is the most reasonable explanation for this
situation?
A) His new bacterial species does not have galE or trpA genes.
B) His new bacterial species cannot survive galE or trpA
mutation.
C) These two genes are too far apart to be mapped by
cotransduction.
D) These two genes are too close together to be mapped by
cotransduction.

84. The reason why it took so long for the gal+ gene to be transferred was
because
A) The gal+ gene was farthest from the origin of transfer.
B) The gal+ gene is the largest gene.
C) The gal+ gene does not readily recombine into the recipient
chromosome.
D) The gal+ gene is susceptible to mutation.