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1. Details of Module and its StructureModule Detail
Subject Name <BOTANY >
Paper Name < Genetics >
Module Name/Title <Microbial Genetics>
Module Id <Module Id>
Pre-requisites <Basic knowledge about bacteria, viruses, fundamentals of genetics, mutations >
Objectives <To brush up student’s knowledge about mutations>
<To make the students aware about the modalities of genetic recombination in bacteria through the process of conjugation>
Keywords <conjugation>,<bacteria>,<F factor>,<mapping>,<genetic recombination>, <mutations>
Structure of Module / Syllabus of a module (Define Topic / Sub-topic of module )
<Topic name1> <Sub-topic Conjugation>, <Sub-topic Conjugation Mapping>
<Topic name2> <Sub-topic Name2.1>, <Sub-topic Name2.2>
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2. 2. Development TeamTABLE OF CONTENTS (for textual content) 1. Introduction
2. Topic 1
2.1 Sub-topic 1: CONJUGATION
2.2 Sub-topic 2: CONJUGATION MAPPING
Role Name Affiliation
National Coordinator < NA >
Subject Coordinator <Dr. Sujata Bhargava> Dept. of Botany, University of Pune, Pune 411 007
Paper Coordinator <Dr. Avinash Kamble> Dept. of Botany, University of Pune, Pune 411 007
Content Writer/Author(CW) <Dr. Anjali Abhay Kulkarni> Dept. of Botany, University of Pune, Pune 411 007
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e-Text
Bacterial Conjugation
General Introduction
Eukaryotes undergo meiosis to reduce the diploid chromosome number of somatic cells to haploid chromosome number of gametic cells. Then the process of fertilization returns these cells to the diploid state of zygote after mixing the genes from two genetically different parents together. Genetic exchanges also take place during these events.
Prokaryotic / Bacterial sexual processes are not so regular. However, they serve the same aim: to mix the genes from two different organisms together and allow exchanges of genetic materials. Such genetic variations are useful for evolution. These can be mediated by any of the three methods:
1. Conjugation: Direct transfer of DNA from one bacterial cell to another, involving direct physical contact. It is driven by the donor cell, via homologous recombination.
2. Transduction: Involves use of a bacteriophage (bacterial virus) to transfer DNA between cells. It is driven by the recipient cell, via homologous recombination.
3. Transformation: Naked DNA is taken up from the environment by bacterial cells. It is driven by the recipient cell.
Much of the study of genetic transfer has been done in E. coli. Some important features of this organism are:
a. It grows readily and rapidly on defined medium that is either solid or liquid. Many generations can be screened for studying genetic effects.
b. It is easily manipulated using standard microbiology techniques due to its small genome size.
c. It can be titered by dilution and plating on solid medium.
d. The cells are haploid and they show large numbers of mutations that can be easily screened.
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Sub-Topic 1: CONJUGATION
1.1 Introduction
Conjugation occurs among both gram-negative and gram-positive bacteria and streptomycetes. Commonly, genes encoding conjugative-transfer functions are associated with an extra-chromosomal replicon, termed as a self-transmissible or conjugative plasmid.
In addition to self-transfer, the transfer systems of conjugative plasmids often facilitate the independent transfer of non-conjugative, mobilizable plasmids that are co-resident in the donor cell. DNA sequences that become cointegrated with the conjugative plasmid can also be transferred. Thus, integration and other recombinational rearrangements can result in transmission of sequences of bacterial chromosome, of transposons, and of other non- mobilizable plasmids. In some cases, even inter-kingdom genetic material transfer is possible. E.g. Transfer of T-DNA from Agrobacterium tumefaciens to plant and mammalian cells (Buchanan-Wollaston, V., Passiatore, J. E., and Cannon, F. (1987) The mob and oriT mobilization functions of a bacterial plasmid promote its transfer to plants. Nature 328:
172–175; Waters V. L. (2001) Conjugation between bacterial and mammalian cells. Nature Genet 29: 375–376) or transfer of plasmids of broad and narrow host range (R751 and F, respectively) from Escherichia coli to Saccharomyces cerevisiae (Heinemann J. A. and Sprague G. F. Jr. (1989) Bacterial conjugative plasmids mobilize DNA transfer between bacteria and yeast. Nature 340: 205–209). Thus conjugation has an immense evolutionary and ecological consequence.
Bacteria that contain transmissible plasmids are called as ‘donor’ or ‘male’ cells and bacteria that receive such plasmids are called as ‘recipient’ or ‘female’ cells. Conjugative plasmids are involved in the dissemination of important traits such as antibiotic resistance, virulence determinants, heavy metal tolerance and metabolic pathways involved in adapting to environmental niches, a process termed as ‘horizontal gene transfer’ (HGT) or
‘lateral gene transfer’ (LGT).
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Conjugation is defined as the process of transferring DNA from a donor to a recipient cell with the establishment of the incoming DNA and its cargo of genetic traits within the trans-conjugant. Conjugation is mediated by self-transmissible plasmids as well as phage-like sequences that have been integrated into the bacterial chromosome, such as integrative and conjugative elements (ICEs) that now include conjugative transposons. Both conjugative plasmids and ICEs can mediate the transfer of mobilizable elements by sharing their conjugative machinery.
Conjugation can either be induced, usually by small molecules or peptides or by excision of the ICE from the host chromosome, or it can be tightly regulated by plasmid- and host- encoded factors. The transfer potential of these transfer regions depends on the integration of many signals in response to environmental and physiological cues.
Maintenance of the conjugative element in the new host/recipient cell is not essential, and the cargo of transferred genes can be incorporated into the recipient cell genome by homologous recombination, transposition or integration.
1.1.1 Initial Experiment 1
Conjugation was first discovered by Lederberg & Tatum in 1946 that involved the F (fertility) factor of E. coli K-12. (Cavalli L. L., Lederberg E. and Lederberg J. M. (1953) An effective factor controlling sex compatibility in Bacterium coli. J. Gen. Microbiol. 8: 89–
103). Refer to Fig. 1.
In this experiment, an auxotrophic E. coli strain with phenotype A (Met-Bio-Thr+Leu+Thi+) was mixed with another auxotrophic phenotype B with complimentary mutations (Met+Bio+Thr-Leu-Thi-). The mixture was plated on minimal agar and it was thought that none of the strains would be able to grow here. But surprisingly, a few colonies developed here and they had a prototrophic phenotype (Met+Bio+Thr+Leu+Thi+) and hence were recombinants. In further continuation of this experiment, strain A was treated with the antibiotic streptomycin and then mixed with strain B. Recombinant colonies formed on the minimal medium but when strain B was treated with the antibiotic streptomycin and then
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mixed with strain A, recombinant colonies were not formed on the minimal medium. This proved that recombinants arose under influence of strain B (donor strain) and strain A was the recipient.
Fig. 1 Lederberg and Tatum experiment showing that sexual recombination occurs between cells of E. coli (Courtesy: Prof. Brooke A. Jude, Bard College, New York)
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1.1.2 Initial Experiment 2
Davis tested whether cell-to-cell contact was required to achieve above mentioned results.
Refer to Fig. 2.
a. Strain A cells were placed on one side of a filter in a U-shaped glass tube, and strain B on the other. One arm of the U-tube was plugged with cotton at its mouth while the other mouth at the other end was connected to a pressure/suction pump. Cells could not move through the filter but molecules moved freely, encouraged by alternating suction and pressure.
b. No prototrophic colonies appeared when the cells were plated on minimal medium.
This indicates that cell-to-cell contact is required, and the genetic recombination results from conjugation.
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Fig. 2 Davis’s U-Tube Experiment (Courtesy: Prof. Brooke A. Jude, Bard College, New York)
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1.2 Structure and Function of Fertility Factor (F Plasmid)
Hayes (1953) showed that genetic transfer in bacteria is unidirectional from donor (F+) to recipient (F-), mediated by a donor sex factor called F.
Fig. 3A
Fig. 3B
FIGURE 3 (A) & (B) Physical and Functional Map of the F Plasmid (100-kb) (Gross and Detailed). Numbers within the map are kilobase coordinates. The transposable elements IS2 (1 copy), 1S3 (2 copies), and Tn1000 (rɗ, 1 copy) are represented by solid boxes. They are important for F integration into recipient chromosomes. The extents of the replication (RepFIA, RepFIB, and RepFIC), transfer, and leading regions are indicated outside the map, as also the origin of conjugative transfer and leading regions. The origin of conjugative transfer (oriT) is denoted by a triangle, indicating the direction of single-stranded DNA transfer (leading region transferred first).
Adapted from: Firth N., Ippen-Ihler K. and Skurray R. A. Structure and Function of the F Factor and Mechanism of Conjugation. Chap. 26
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‘Fertility’ or ‘F’ plasmid (Fig. 3A and 3B) is an extra-chromosomal supercoiled, circular DNA molecule with mass of 62x106 and length 30 µm. It is also known as an episome. It is approximately 100 kb in size with 100 genes. It has 1-2 copies per cell and is self- transmissible. It contains genes coding for its own replication and maintenance within the bacterial cell. It also codes for proteins involved in conjugation, like pilin protein synthesis and aggregate stabilization. It can grow autonomously within the bacterial cell or may get stably integrated into the main bacterial chromosome forming Hfr (High Frequency Recombinant) donor cells. This integration is a reciprocal DNA exchange, occurring at more than 20 sites on bacterial chromosome, with variable frequencies. The integrated F sequence is always flanked by two copies (direct repeats) of one of the transposable IS (Insertion Sequence) elements of the F plasmid. F exchange occurs predominantly in IS 3, followed by IS 2, IS 1 and Tn 1000 (rɗ). F plasmid can get integrated in either clockwise or anticlockwise directions, depending on orientation of IS elements on the bacterial chromosome.
F factor bears genes for replication control (incompatibility) and hence appears to inhibit the replication and establishment of additional F and F-like plasmids that may enter the same cell. But the bacterial cell may harbor additional non-F plasmids suggesting that many replication sites are available in the bacterial host.
1.2.1 Organization of F-Plasmid (Refer to Figs. 3A & 3B)
1) Leading Region
The F DNA sequences located between the origin of conjugal transfer, oriT, and autonomous replication, RepFIA are presumed to be the first to enter the recipient cell during conjugation and hence are designated the ‘leading region’. Leading-region gene products are thought to assist in establishing F DNA in the recipient. Leading region also contains Transposons and TRA or transfer region involved in development of Pili.
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2) Autonomous Replication
The RepFIA region, believed to be primarily responsible for the typical replication properties of F, contains both unidirectional (oriS) and bidirectional (oriV) replication origins. Stringent regulation of RepFIA and associated maintenance and partitioning mechanisms act in concert to sustain the F plasmid at one to two copies per cell. The secondary replication region, RepFIB, is independently functional and can sustain plasmid replication in the absence of RepFIA. The RepFIC region includes an incomplete remnant of a replication system that is used by some other related plasmids.
3) Transposable Elements
The F sequence includes a single copy each of Tn1000 (also known as rɗ) and IS2 and two copies of IS3 (Fig. 3A). Tn1000 (rɗ) appears to have interrupted RepFIC, whereas IS3 inactivation of the transfer region regulatory gene, finO, is responsible for the constitutively high levels of conjugative transfer exhibited by F. These elements also mediate the F- chromosomal integration events that form Hfr donors which, through imprecise excision, can subsequently generate F-prime (F’) plasmids. The insertion of transposable elements into an F-plasmid progenitor was fortuitous for the efficient transfer of chromosomal markers crucial to the original detection of conjugation and to the subsequent utility of F.
4) Conjugative Transfer
Including the oriT site (map position 66.7F) and extending to the HindIII restriction cleavage site in IS3 (coordinate 100/0F), the transfer (tra) region encodes all of the F loci known to be required for efficient conjugative transfer; its 33.3-kb nucleotide sequence has recently been compiled.
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1.3 F-Conjugation Process
1) Cell-to-Cell Contact
F+ and Hfr cells produce a unique surface component that enables them to form a union with F– cells (‘Effective Contact’). Contact between F+ donor and F– recipient cells is believed to be instigated by an interaction between the tip of an F pilus and the recipient cell surface. When F+ donor and F– recipient cells are separated by a porous membrane, recombinants were not produced. Thus although genetic material could move across the membrane, recombination does not occur. Conjugating cells are typically aggregated in close wall-wall association. F DNA transfer processes are believed to be precipitated by a
“mating signal” generated by functional-pair formation. A significant number of chromosomal genes aid in conjugation by encoding pilus components, as well as proteins involved in expression and biogenesis of pilus. A pilus is a hair-like protein appendage connecting donor and recipient bacterial cells (Fig. 6). 1.5 - 2.7 F piIi can be produced per cell depending on the growth medium. Each pilus contains a single, hydrophobic protein
‘pilin’ forming a hollow tubular structure. It is postulated that the pilus first brings donor and recipient cells into initial contact at pilus tip and subsequently allows very close wall-to-wall contact between the cells (Helmuth R., and Achtman M. 1978. Cell-cell interactions in conjugating Escherichia coli: purification of F pili with biological activity. Proc. Natl. Acad.
Sci. USA 75: 1237–1241). In thin sections, the cells exhibit relatively large, electron-dense regions of envelope association, termed conjugative junctions (Fig. 4A and B). These are believed to generate a ‘mating signal’ to precipitate F DNA transfer processes (Kingsman A., and Willetts N. 1978. The requirements for conjugal DNA synthesis in the donor strain during Flac transfer. J. Mol. Biol. 122: 287–300). Once these junctions are formed, the pilin proteins are depolymerized and pili are retracted back into the donor cell envelopes.
These donor and recipient contacts then become stabilized in a manner that renders the aggregate more resistant to shear forces.
When DNA transfer is completed, mating cells actively disaggregate to yield two cells capable of donor activity.
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Pilus Initiation Pilus Binding Pilus Retraction Stabilization
FIGURE 4 (A) & (B) Stages of F-mediated Conjugal DNA Transfer From: Firth N., Ippen-Ihler K.
and Skurray R. A. Structure and Function of the F Factor and Mechanism of Conjugation. Chap. 26
(A) Intercellular contact begins when tip of F pilus is extended from F+ donor cell to F- recipient cell. Depolymerization of filament subunits brings the cell surfaces together and they get stabilized due to specific surface interactions. The electron dense ‘conjugative junction’ is shown by hatched lines.
(B) DNA transfer begins when the pilus transmits a ‘mating signal’ to a protein complex including traI product at F-oriT site (Red arrow in the Fig. B). This stimulates displacement of a single strand of F DNA that is passed in the recipient cell. After nicking, the F-TraI relaxase/Helicase remains attached to the 5’ oriT end ate the site of intercellular connection. As the strand is unwound from oriT in the 5’ 3’ direction and moved in the recipient cell, replacement strand synthesis in the donor and complementary strand synthesis in the recipient also occurs.
Transfer terminates when the transferred strand is again recircularized at oriT and the two daughter cells can now separate, with both becoming F+ donor cells.
B
A
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DNA-related Events Necessary for Conjugative Transfer
1. Transfer systems utilize type IV secretion systems (T4SS), which form the transferosome, to transport the DNA between cells.
2. Briefly, a protein complex (oriT complex) is associated with the origin of transfer of transmissible and mobilizable plasmids. One DNA strand in the oriT site is “nicked”
by a nucleoprotein complex: relaxosome that contains the enzyme relaxase catalyzing the single strand DNA ‘nick’ and subsequent covalent attachment of the 5’ end of the DNA to the protein; as well as auxiliary proteins bound to adjacent sites. The F-plasmid relaxase, TraI, is also a helicase and can unwind the nicked strand in the 5’ to 3’ direction. This enzyme, a phosphor-di-ester transferase, is part of the RCR (Rolling Circle Replication) system of the bacteria (Llosa M., Gomis- Rüth F. X., Coll M. and de la Cruz F. (2002) Bacterial conjugation: a two-step mechanism for DNA transport. Mol. Microbiol. 45: 1–8).
3. The relaxosome is then linked to the transferosome at membrane via a unique
‘coupling protein’. Mobilizable elements encode an oriT region and the components of the relaxosome but use the transferosome and coupling protein (usually) to affect their transfer (Fig. 4B) (de la Cruz F., Frost L. S., Meyer R. J. and Zechner E. L. (2009) Conjugative DNA metabolism in Gram-negative bacteria.
FEMS Rev. Microbiol. 34: 18–40).
4. The coupling protein is also shown to act as an adaptor molecule to tailor the donor DNA strand of F plasmid to the T4SS geared for protein transport (Llosa M., Gomis- Rüth F. S., Coll M. and de la Cruz F. (2002) Bacterial conjugation: a two-step mechanism for DNA transport. Mol. Microbiol. 45: 1–8). During transfer, this protein is suggested to be associated with the site of intercellular connection through which the single strand of DNA is passed; because it is attached to the 5’ end of oriT and works as a pushing molecular motor, conferring processivity to the secretion
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process. It may also catalyse re-circularization of the transported strand to terminate transfer.
5. Transfer of single stranded DNA from donor to recipient cell occurs by the RCR mechanism, initiating at 5’ end. Energy required for this transfer is thought to be contributed by TraI protein while TraD protein contributes to actual transport of single stranded DNA from donor cell to recipient cell across the membranes.
Subsequently, the transferred strand is converted into a circular double stranded molecule in the recipient cell, even before the F DNA transfer is completed.
6. Replacement strand synthesis in the donor and complementary-strand synthesis in the recipient depend on host enzymes (traM and traD gene products) and are not essential for DNA transfer per se. Figure 5 shows synthesis in the donor by a rolling-circle mechanism. A variation in which both the 5’ and 3’ oriT ends remain associated with TraI has also been suggested. The F-TraI protein forms a covalent linkage only with the 5’ oriT end; whether the 3’ end remains bound in some other persistent association is not yet clear.
7. F plasmid transfer is negatively affected by temperature, late stationery phase of culture growth, reduced cyclic AMP levels etc.
2) Completion of Conjugative Transfer
1. Completion of transfer has been proposed to depend on recognition of the oriT site reconstituted by replacement strand synthesis, cleavage, and ligation of the newly generated 3’ end to the 5’ end of the transferred strand. The sequence required for termination extends no more than 36 bases to the right of the nick site and includes phased poly(A) tracts which specify a sequence-determined bend (Gao Q., Luo Y.
and Deonier R. C. (1994) Initiation and termination of DNA transfer at F plasmid oriT. Mol. Microbiol. 11: 449–458).
2. When DNA transfer is completed, mating cells actively disaggregate to yield two cells capable of donor activity.
3. traS and traT genes encode proteins responsible for ‘surface exclusion’ that limit host cell capacity to act as recipient for same or closely-related plasmids. The
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expression of surface exclusion is probably a fundamental requisite of donor ability, since in its absence; donor cell populations would bear the metabolic cost of continuous and futile recipient-donor activity (Firth N., Ippen-Ihler K. and Skurray R.
A. Structure and Function of the F Factor and Mechanism of Conjugation. Chap. 26 and references therein).
4. Kado and his colleagues not only identified amino acid sequence similarity between pilin proteins and VirB products believed to form an envelope spanning mating structure that facilitates agrobacterial T-DNA transfer to plant cells, but also demonstrated that protein VirB2 is processed in an analogous fashion to F propilin (Shirasu K., Koukolikova-Nicola Z., Hohn B., and Kado C. I. (1994) An inner- membrane associated virulence protein essential for T-DNA transfer from Agrobacterium tumefaciens to plants exhibits ATPase activity and similarities to conjugative transfer genes. Mol. Microbiol. 11: 581–588).
.
FIGURE 5 Diagram of the main steps and components in the conjugation process in Gram- negative bacteria. From: Frost L. S. and Koraimann G. Regulation of bacterial conjugation:
balancing opportunity with adversity. Future Microbiol. (2010) 5: 1057– 1071.
The inset shows the main components of the transfer apparatus. All conjugative systems require a coupling protein, an ATPase that energizes transfer. Most Gram-negative and -positive conjugative
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systems require a T4SS, which forms the conjugative pore, and a relaxosome composed of the relaxase, which nicks at oriT and other auxiliary proteins. Gram-negative systems, but not Gram- positive ones, produce an extracellular appendage called a pilus, which identifies a suitable recipient cell. Some systems, such as in Streptomyces, require only a coupling protein for DNA transfer.
Main diagram: The donor cell in blue binds the recipient cell in red via the pilus, which retracts and brings the cells together. A signal is transmitted through the T4SS (Type 4 Secretion System) to the coupling protein that then attracts the relaxosome to the conjugative pore. A single strand of DNA, covalently linked to the relaxase, is transported into the recipient cell, followed by the establishment and replication of the plasmid, or in the case of an integrating conjugative element, incorporation into the recipient chromosome. Recipient cells that are newly converted to donor cell status exhibit high frequency of transfer, which can lead to epidemic spread. Many control elements alter the level of transfer gene expression in the donor cell or interfere with the transfer and establishment of the plasmid in the recipient. The major negative influences are indicated in red boxes whereas the steps in conjugation aand establishment are indicated in green boxes. (IM:
Inner membrane; OM: Outer membrane.)
FIGURE 6 An Electromicrograph of Two Conjugating Bacteria Showing Pili (red arrows) Between Them (Courtesy: Prof. Brooke A. Jude, Bard College, New York).
Pili
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1.4Integration of Plasmid DNA into Bacterial Chromosome
Cavalli and Hayes (Strickberger M. W., ‘Genetics’, Publ. Macmillan Publishing Company, New York, 20 ). discovered that some F+ populations of E. coli gave rise to donor strains which could transmit chromosomal genes in high frequencies (One per thousand cells vis- à-vis one per million cells of normal F+ plasmid). These strains are called as as ‘High Frequency Recombinant’ (Hfr) Strain. It was found that for these strains, genes encoded on the F factor can integrate into the host chromosomal DNA, and alter its genotype / phenotype. Also bacterial chromosomal DNA becomes bigger in such cases. An Hfr strain is derived from an F+ strain, by default.
1. Integration of F factor DNA into the host chromosomal DNA occurs at more than 20 different homologous locations, by reciprocal DNA exchange. F-DNA can integrate either in a clockwise or in an anticlockwise direction depending on the orientation of IS elements on the chromosome.
2. The integrated F-DNA Seq. is always flanked by two copies of direct repeats of one of the transposable IS (Insertion Sequence) elements found both in the F plasmid and Chromosomal DNA. Maximum numbers of reciprocal exchanges occur at IS3, followed by IS2, IS1 and ɤɗ.
3. Hfr strain transfers chromosomal DNA to F- strains in a directional manner. This transfer begins at the origin of transfer and is completed within 100 minutes. The Hfr cell tries to transfer the entire bacterial chromosome to the F- cell and hence the first DNA to be transferred is chromosomal DNA, and the last DNA to be transferred will be the F factor DNA.
4. The amount of DNA transferred depends on the duration of conjugation. In most cases, the recombinants produced by Hfr strains remain F- and only a few inherit the Hfr state, where the conjugation has taken place for quite some duration. Fig. 7 pictorially depicts formation of Hfr strains.
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FIGURE 7 Formation of HFr Strains
5. Sometimes the F-DNA integrated in the bacterial chromosome is excised imperfectly such that a few bacterial genes are excised along with F-DNA. This gives rise to F’ Plasmids (Fig. 8). An F’ plasmid is named for the gene(s) it carries, e.g., F’ (lac).
6. Here the excision involves a different bacterial IS element than was originally used for integration. These ‘Substituted F Factors’ (F’) were first described by Adelberg
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and Burns (Strickberger M. W., ‘Genetics’, Publ. Macmillan Publishing Company, New York, 20 ).
7. In all of these cases, due to transmission of donor cell chromosome material, recombination with recipient chromosome material occurs giving rise to new gene combinations.
Because of the chromosomal DNA, F’ can cause ‘F-duction’ or ‘sex-duction’ for the specific genes carried. E.g. Male donor cells with F’lac+ will preferentially transmit this gene to F-lac- recipients, converting them to lac+. This is the most preferential combination of recombinants observed. Thus ‘F-duction’ or ‘sex-duction’ is defined as the transmission of bacterial genes to a recipient cell through their incorporation into an autonomous conjugative plasmid.
Fig. 8 Formation of F’ Factor
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Sub-Topic 2: CONJUGATION MAPPING
Since pili are delicate structures, disruption of conjugation / mating can occur even with a slight interference. The chances of breakage can occur randomly throughout the length of the chromosome, so that partial transfer of donor chromosome is observed. These properties enabled the development of a mapping procedure for bacterial chromosomal DNA, called as ‘Conjugation Mapping’.
Hfr strains showed a gradation in frequency in which donor loci appeared among the recombination products, when conjugation was permitted for different time intervals.
Consider the following cross:
Hfr (A+B+C+………..Y+Z+) X F- (A-B-C-………..Y-Z-)
A+ gene appeared most frequently, J+ less frequently and Z+ least frequently; as if determined by order of their entry. If the recombinant inherits the Hfr state, it shows recombination for genes with the lowest frequency e.g. Z+ in above case. Thus Hfr factor appears to be connected to the terminal end of the bacterial chromosome. This also offers the opportunity for mapping the gene sequence in the donor chromosome by timing the entry of different genes. Hence Jacob and Wollman (1950s) developed the ‘interrupted mating technique’. This is depicted pictorially in Fig. 9.
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FIGURE 9 Interrupted Mating Technique (Courtesy: Prof. Brook A. Jude)
2.1 Steps in Interrupted Mating and Mapping (Figs. 9, 10A, 10B) – Experiment by Jacob and Wollman (1958)
Mix cultures of E. coli strain HfrH (prototrophic, thr+leu+aziRtonRlac+gal+strR) and F- (thr-leu-aziStonSlac-gal-strS), permitting them to conjugate for a short period of time at 37°C.
Samples are removed at different time points and agitated in a Waring Blendor to separate conjugating pairs and to break donor chromosomal exchange. Selective media are used to analyze the transconjugants.
Plate the cells on a medium with streptomycin but lacking threonine. This medium selectively killed (counter-selected) the HfrH donors and permitted the growth of
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(selected) only those recombinants / F- recipients that had incorporated thr+ gene.
Incidentally thr was found to be the first gene to enter into the recipients and after 8.5 minutes, when conjugation was interrupted, thr+ recipients were obtained.
Thus for HfrH strain, transfer begins at thr locus. The strain is prototrophic, able to grow on minimal media but streptomycin sensitive. F- recipient is auxotrophic for threonine (thr-), that is requires threonine supplementation to minimal medium for growth, sensitive to sodium azide (azi-) and to bacteriophage T1 (ton A) and unable to ferment lactose (lac-) and galactose (gal-) but resistant to streptomycin. Here streptomycin acts as a selective marker that helps to eliminate any non- recombinants and donor cells after termination of conjugation.
At least an 8-minute time interval after conjugation was found to be necessary for chromosomal transfer to begin.
Subsequently thr+ and strr recombinants were selected and tested for the presence of other donor genes (Unselected Markers) after longer time periods of conjugation.
It was found that as the length of time a mating occurs increases, more amount of DNA is transferred.
By mating for different times, you can get DNA of several sizes, and determine the order of the genes, and how far apart they are, in terms of time units. Here 1 time unit = 1 minute.
After about 9 min. of mating, azi gene was found to enter the recipient and by 20 min. of mating 90% of the recombinants were thr+azi+.
This was followed by entry of ton (10 min.), lac (17 min.) and gal (25 min.) genes.
But it was observed that even after long periods of mating, a gene located relatively far from the origin (such as gal) had less chance of being incorporated into the recipient chromosome even if there was sufficient time for it to have entered the cell.
This showed that the Hfr DNA appears to be transferred in a linear manner.
Different Hfr strains initiate transfer of bacterial chromosome at different points and hence linkage maps can be established based on different recombination frequencies of various loci (Fig. 10 (A) & (B)).
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FIGURE 10 A Conjugation Mapping (Diagrammatic)
FIGURE 10 B Appearance of Donor Genetic Markers in Recipient over Time (Modified from Peter J. Russell, I-Genetics, Chapter 14)
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Now the entire E. coli chromosome has been mapped to a complete length of 100 time units. Here the relationship between genes is more easily determined as a function of time rather than as a function of recombination frequencies. For genes that are more than 3 time units apart, time unit mapping offers a precise estimate of the linkage order but for genes closer together, multiple mating experiments have to be done to determine the precise linkage order.
FIGURE 11 Full Genetic Map of E. coli K12 Strain H – Here the mutations are mapped in time units (inner circumference). thr can be seen at 0 min. Out of 1000 gene loci identified, only a few are shown here for the sake of clarity (Courtesy: Prof. Brook A. Jude).
