GM Unit 1.pptx

1. 1
Microbial Genetics
Unit 1
Dr. Krishan Kumar
Assistant professor
Department of Biotechnology
Parul Institute of Applied Sciences

2. The transfer of genetic information by direct cell to cell contact
• Joshua Lederberg and Edward L. Tatum in 1946 performed an experiment. They mixed
two auxotrophic strains, incubated the culture for several hours in nutrient medium, and
then plated it on minimal medium.
• For example, one strain required biotin (Bio‾), phenylalanine (Phe‾), and cysteine (Cys‾)
for growth, and another needed threonine (Thr‾), leucine (Leu‾), and thiamine (Thi‾).
• Recombinant prototrophic colonies appeared on the minimal medium after incubation.
Thus the chromosomes of the two auxotrophs were able to associate and undergo
recombination
2
Bacterial Conjugation
Prototrophic microorganisms can synthesize all the nutrients that are required for their growth from minimal medium
without the addition of supplements. However, many microorganisms in nature (and in culture collections) are
auxotrophic; that is, they are unable to synthesize all of the vital nutrients.

3. 3
Lederberg and Tatum experiments
Figure 13.12 Evidence for Bacterial Conjugation.
Lederberg and Tatum’s demonstration of genetic
recombination using triple auxotrophs.

4. 4
Bernard Davis U-Tube Experiment
• Lederberg and Tatum did not directly prove that physical contact of the cells was
necessary for gene transfer.
• This evidence was provided by Bernard Davis (1950), who constructed a U tube
consisting of two pieces of curved glass tubing fused at the base to form a U shape with a
fritted glass filter between the halves. The filter allows the passage of media but not
bacteria.
• The U tube was filled with nutrient medium and each side inoculated with a different
auxotrophic strain of E. coli (figure 13.13). During incubation, the medium was pumped
back and forth through the filter to ensure medium exchange between the halves.
• After a 4 hour incubation, the bacteria were plated on minimal medium. Davis discovered
that when the two auxotrophic strains were separated from each other by the fine filter,
gene transfer could not take place. Therefore direct contact was required for the
recombination that Lederberg and Tatum had observed.

5. 5
Bernard Davis U-Tube Experiment
Figure 13.13 The U-Tube Experiment.
The U-tube experiment used to show that
genetic recombination by conjugation requires
direct physical contact between bacteria.

6. 6
William Hayes demonstration
• In 1952 William Hayes (1913-1994) demonstrated that the gene transfer
observed by Lederberg and Tatum was unidirectional. That is, there were
definite donor (F+, or fertile) and recipient (F-, or nonfertile) strains, and
gene transfer was nonreciprocal.
• He also found that in F+ X F- mating, the
progeny were only rarely changed with
regard to auxotrophy (i.e., chromosomal
genes usually were not transferred).
However, F- strains frequently became F+.
• This result was further explained and it
was found that F+ strains carried a
plasmid which was known as F factor. F
factor carrying the genes for sex pilus
formation and plasmid transfer.

7. 7
Figure 10.16 Genetic map of the F (fertility) plasmid of
Escherichia coli. The numbers on the interior show the size in
kilobase pairs (the exact size is 99,159 bp). The region in dark
green at the bottom of the map contains genes primarily
responsible for the replication and segregation of the F
plasmid. The origin of vegetative replication is oriV. The light
green tra region contains the genes needed for conjugative
transfer. The origin of transfer during conjugation is oriT. The
arrow indicates the direction of transfer (the tra region is
transferred last). Insertion sequences are shown in yellow.
These may recombine with identical elements on the bacterial
chromosome, which leads to integration and the formation of
different Hfr strains.
F factor plasmid
Perhaps the best-studied conjugative plasmid is F factor. It plays a major role in conjugation
in E. coli, and it was the first conjugative plasmid to be described.

8. 8
F factor plasmid
Perhaps the best-studied conjugative plasmid is F factor. It plays a major role in conjugation
in E. coli, and it was the first conjugative plasmid to be described.
Figure 16.16: The F plasmid. Transfer (tra) genes are shown in red, and some of their functions are indicated. The plasmid also
contains three insertion sequences and a transposon. The site for initiation of rolling-circle replication and gene transfer
during conjugation is oriT.

9. 9
• The F factor is about 100,000 bases long and bears genes responsible for cell
attachment and plasmid transfer between specific E. coli cells.
• Most of the information required for plasmid transfer is located in the tra operon,
which contains at least 28 genes. Many of these direct the formation of sex pili that
attach the F+ cell (the donor cell containing an F plasmid) to an F- cell. Other gene
products aid DNA transfer.
• In addition, the F factor has several IS elements that assist plasmid integration into the
host cell's chromosome. Thus the F factor is an episome that can exist outside the
bacterial chromosome or be integrated into it
Figure 16.17 Bacterial Conjugation. An electron
micrograph of two E coli cells in an early stage
of conjugation. The F+ cell to the right is
covered with fimbriae, and a sex pilus connects
the two cells.

10. 10
Figure 16.18 F Plasmid Integration.
The reversible integration of an F plasmid
or factor into a host bacterial chromosome.
The process begins with association
between plasmid and bacterial insertion
sequences. The 0 arrowhead (white)
indicates the site at which oriented transfer
of chromosome to the recipient cell begins.
A, B, 1, and 2 represent genetic markers.

11. 11
F+ x F- Mating
• In 1952 William Hayes (1913-1994) demonstrated that the gene transfer observed by
Lederberg and Tatum was unidirectional. That is, there were definite donor (F+, or
fertile) and recipient (F-, or nonfertile) strains, and gene transfer was
nonreciprocal.
• He also found that in F+ X F- mating, the progeny were only rarely changed with
regard to auxotrophy (i.e., chromosomal genes usually were not transferred).
However, F- strains frequently became F+.
• This result was further explained and it was found that F+ strains carried a plasmid
which was known as F factor. F factor carrying the genes for sex pilus formation
and plasmid transfer.
• The sex pilus is used to establish contact between the F+ and F- cells. Once contact is
made, the pilus retracts by disassembling its subunits. This bringing the cells
into close physical contact.
• The F+ cell prepares for DNA transfer by assembling a type IV secretion apparatus,
using many of the same genes used for sex pilus biogenesis. The F factor then
replicates by rolling-circle replication.

12. 12
• During rolling-circle replication, one strand of the circular DNA is nicked,
and the free 3' -hydroxyl end is extended by replication enzymes. The 3' end
is lengthened while the growing point rolls around the circular template and
the 5' end of the strand is displaced to form an ever-lengthening tail. The
single-stranded tail may be converted to the double-stranded form by
complementary strand synthesis.
• During conjugation, rolling-circle replication is initiated by a complex of
proteins called the relaxosome, which are encoded by the F factor. The
relaxosome nicks one strand of the F factor at a site called oriT (for origin of
transfer). Relaxase, an enzyme associated with the relaxosome, remains
attached to the 5' end of the nicked strand.
• As F factor is replicated, the displaced strand and the attached relaxase
enzyme move through the type IV secretion system to the recipient cell.
During plasmid transfer, the entering strand is copied to produce double-
stranded DNA. When this is completed, the F recipient cell becomes F+.

13. 13

14. 14
Hfr Conjugation
• By definition, an F+ cell has the F factor free from the chromosome, so in an
F+ X F- mating, chromosomal DNA is not transferred.
• As F factor is an episome plasmid, It can integrate into chromosome under
certain circumstances. Cells with an F plasmid integrated into the
chromosome are called Hfr cells (Because of the high frequency of
recombinants produced by this mating, it is referred to as Hfr conjugation
and the donor is called an Hfr strain).
• When integrated, the F plasmid's tra operon is still functional; the plasmid
can direct the synthesis of pili, carry out rolling-circle replication, and
transfer genetic material to an F- recipient cell.
• If the cells remain connected, the entire chromosome with the rest of the
integrated F factor will be transferred; this takes about 100 minutes to
accomplish.

15. 15
• However, the connection between the cells usually breaks before this
process is finished. Due to conjugation break, a part of chromosome DNA
along with part of Plasmid DNA is transferred in to the recipient cell. Thus
a complete F factor is rarely transferred, and the recipient remains F-.
• After the replicated donor chromosome enters the recipient cell, it may be
degraded or incorporated into the F- genome by recombination.
Figure 10.20 Transfer of chromosomal genes by an Hfr
strain. The Hfr chromosome breaks at the origin of
transfer within the integrated F plasmid. The transfer
of DNA to the recipient begins at this point.

16. 16
Figure 16.22 Creation of Hfr Strains and Hfr x
F- Conjugation.
(a) Integration of the F factor into the donor cell's
chromosome creates an Hfr cell. (b) During Hfr X
F- conjugation, some plasmid genes and some
chromosomal genes are transferred to the
recipient. Note that only a portion of the F factor
moves into the recipient. Because the entire
plasmid is not transferred, the recipient remains F-
. In addition, the incoming DNA must recombine
into the recipient's chromosome if it is to be
stably maintained.

17. 17
F' Conjugation
Because the F plasmid is an episome, it can leave the bacterial chromosome and resume
status as an autonomous F factor. Sometimes during excision an error occurs and a portion of
the chromosome is excised, becoming part of the F plasmid.
Because this erroneously excised plasmid is larger and genotypically distinct from the
original F factor, it is called an F' plasmid.
A cell containing an F' plasmid retains all of its genes, although some of them are on the
plasmid. It mates only with an F- recipient, and F' X F- conjugation is similar to an F+ X F-
mating.
The recipient becomes F' and is partially diploid because the same bacterial genes present on
the F' plasmid are also found on the recipient's chromosome. In this way, specific bacterial
genes may spread rapidly throughout a bacterial population.

18. 18
Figure 16.23 F' Conjugation.
(a) Due to an error in excision, the A gene of an Hfr
cell is picked up by the F factor. (b) During
conjugation, the A gene is transferred to a recipient,
which becomes diploid for that gene (i.e., Aa).

19. 19
Interrupted mating and temporal mapping
• The F plasmid and the chromosome of E. coli, both carry several copies of
mobile elements called insertion sequences. These provide regions of
sequence homology between chromosomal and F plasmid DNA.
Consequently, homologous recombination between an IS on the F plasmid
and a corresponding IS on the chromosome results in integration of the F
plasmid into the host chromosome.
• Because several distinct insertion sequences are present on the chromosome,
a number of distinct Hfr strains are possible. A given Hfr strain always
donates genes in the same order, beginning at the same position. However,
Hfr strains that differ in the chromosomal integration site of the F plasmid
transfer genes in different orders (Figure). At some insertion sites, the F
plasmid is integrated with its origin pointing in one direction, whereas at
other sites the origin points in the opposite direction.

20. 20
• The orientation of the F plasmid determines which chromosomal genes
enter the recipient first. By using various Hfr strains in mating
experiments, it was possible to determine the arrangement and
orientation of most of the genes in the E. coli chromosome long before it
was sequenced.
Figure 10.22 Formation of different Hfr strains.
Different Hfr strains donate genes in different
orders and from different origins.
(a)F plasmids can be inserted into various insertion
sequences on the bacterial chromosome, forming
different Hfr strains.
(b)(b) Order of gene transfer for different Hfr
strains.

21. 21

22. 22

23. 23

24. 24

25. 25

26. 26
1. What is bacterial conjugation and how was it discovered?
2. F Factor map.
3. For F+, Hfr, and F- strains of E. coli, indicate which acts as a donor during
conjugation, which acts as a recipient, and which transfers chromosomal
DNA.
4. Describe how F+ X F- and Hfr conjugation processes proceed, and
distinguish between the two in terms of mechanism and the final results.
5. Compare and contrast F+x F- and F' X F- conjugation.
6. Interrupted mating and temporal mapping
IMP

27. 27
Transposable genetic elements
• Short sequence of DNA that has ability to move from one location to
another location within genome. These elements are often referred to as "
jumping genes," mobile genetic elements , and transposable elements.
• Transposition refers to the movement of a mobile genetic element.
• Mobile genetic elements were first discovered in the 1940s by Barbara
McClintock (1902-1992) during her studies on maize genetics (a discovery
for which she was awarded the Nobel Prize in 1983).
• The enzymes that function in transposition are collectively termed
recombinases. However, the recombinase used by a specific mobile genetic
element may be called an integrase, resolvase, or transposase.
• The simplest mobile genetic elements in bacteria are insertion sequences,
or IS elements for short

28. 28
IS elements
• An IS element is a short sequence of DNA (around 750 to 1,600 base pairs
[bp] in length). It contains only the gene for the enzyme transposase, and
it is bounded at both ends by inverted repeats-identical or very similar
sequences of nucleotides in reversed orientation.
• Inverted repeats are usually about 15 to 25 base pairs long and vary among
IS elements so that each type of IS has its own characteristic inverted
repeats.
• Transposase is required for transposition and accurately recognizes the
ends of the IS. Each IS element is named by giving it the prefix IS followed
by a number. IS elements have been observed in a variety of bacteria and
some archaea.

29. 29
Figure 16.13 Transposable Elements.
All transposable elements contain common
features. These include inverted repeats (IRs)
at the ends of the element and a recombinase
(e.g., transposase) gene. (a) Insertion
sequences (IS) consist only of IRs on either
side of the transposase gene. (b) Composite
transposons and (c) unit transposons contain
additional genes (e.g., antibiotic-resistance
genes) in addition to the recombinases that
enable them to transpose. In composite
transposons, the additional genes are flanked
by insertion sequences, which supply the
transposase. Unit transposons are not
associated with insertion sequences. DRs,
direct repeats in host DNA, flank a
transposable element.

30. 30
Transposons
• Transposons are more complex in structure than IS elements.
• Some transposons (composite transposons) consist of a central region
containing genes unrelated to transposition (e.g., antibiotic-resistance
genes) flanked on both sides by IS elements that are identical or very
similar in sequence (figure 16.13b). The flanking IS elements encode the
transposase used by the transposon to move.
• Other transposons (Unit Transposon) lack IS elements and encode their
own transposition enzymes (figure 16.13c). Most transposon names begin
with the prefix Tn.
• Some transposons bear transfer genes and can move between bacteria
through the process of conjugation. They are called conjugative transposons
or integrative conjugative elements (ICEs). A well-studied example of an
ICE is Tn916 from Enterococcus faecalis.

31. 31

32. 32
Transposition methods
Two major transposition methods have been identified: simple transposition
and replicative transposition (figure 16.14).
Simple transposition is also called cut-and-paste transposition. In this
method, transposase catalyzes excision of the transposable element, followed
by cleavage of a new target site and ligation of the element into this site.
Target sites are specific sequences about five to nine base pairs long. When a
mobile genetic element inserts at a target site, the target sequence is
duplicated so that short, direct-sequence repeats flank the element's terminal
inverted repeats.
In replicative transposition, the original transposon remains at the parental
site on the chromosome and a copy is inserted at the target DNA site (figure
16.14b).

33. 33

34. 34

35. 35
1. How does a transposon differ from an insertion
sequence?
2. What is simple (cut-and-paste) transposition? What is
replicative transposition? How do the two mechanisms
of transposition differ? What happens to the target site
during transposition?
Transposition Question