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Gene transfer
Vertical gene transfer: genes from mother to daughter cell or from parents to offspring Horizontal gene transfer (HGT): i.e. Lateral gene transfer (LGT) genetic material from one organism is passed on to another directly, outside the sexual reproduction route.

Binary Fission

Filamentous temperature sensitive sensitive



Superbug: MRSA
Methicillin Resistant Staphylococcus Aureus y library/news/080401_mrsa /may/22/ global

MRSA bacteria
magnified x9560


Transfer of genetic material from one bacterium to another can occur in three ways:
Involves direct physical contact

I Involves viruses l i

Involves uptake from the environment

Bacterial conjugation is the transfer of genetic material between bacteria through direct cell-to-cell contact J h Lederberg and Edward Tatum (1946) Joshua d b d Ed dT
They were studying strains of Escherichia coli that y y g had different nutritional growth requirements
One strain was designated bio met phe+ thr+ g p
It required one vitamin (biotin) and one amino acid (methionine) It could produce the amino acids phenylalanine and threonine

The other strain was designated bio+ met+ phe thr

Had the opposite requirements for growth

Genetic material was transferred between the two strains

Physical contact between the bacterial strains was required

No colonies

Nutrient agar plates lacking biotin, methionine, phenylalanine and threonine

No colonies

Sex pili
F+ (donor) strain forms sex pili
Slender hollow structures on the surface of the bacterial cell Act as attachment sites that promote binding between bacteria F+ strain makes physical contact with F- strain Contact stimulates donor cell to begin genetic transfer

Who are the gene donors?

Conjugative plasmids
e.g., F factors These plasmids carry genes required for conjugation e g conjugation, e.g., Gene for pilin protein is necessary for the production of a sex pilus

oriT F+ strain

"Hfr" strain

Process of conjugation
Plasmid encoded protein complex Plasmid-encoded (relaxosome) cuts one strand of plasmid DNA at the origin of transfer
Cut strand of DNA is termed T-DNA This strand will be transferred to the recipient cell

R l Relaxosome separates DNA strands t t d of plasmid

Process of conjugation
T DNA/relaxase complex is T-DNA/relaxase recognized by a coupling factor Coupling factor promotes entry of this complex into the exporter
Complex of 10 15 plasmid plasmidencoded proteins Spans bacterial membrane(s)

T-DNA/relaxase complex is pumped through pilus into recipient cell i i t ll

Other strand remains in donor cell

Process of conjugation

Relaxase circularizes T-DNA in recipient cell DNA replication converts DNA in donor and recipient cells to double-stranded d bl t d d condition diti Results of conjugation
Donor cell is unchanged Recipient cell has become F+

Hfr Strains
Some strains are very efficient at transferring many chromosomal genes to recipient F- strains
Designated Hfr strains
High frequency of recombination

F factor has become integrated into the bacterial chromosome

An episome is a segment of DNA that can exist as a plasmid and integrate into the chromosome

Conjugation between an Hfr strain and an F- strain j g f

Chromosomal genes nearest the origin are transmitted most efficiently y Likelihood of transfer decreases with distance from origin g

Animation http://highered mcgraw hill com/sites/0072835125/student view0/index html

Interrupted Mating Technique

A technique used to map bacterial genes by determining the sequence in which donor genes enter recipient cells. A gene mapping technique in which bacterial conjugation is disrupted after specified time intervals. The Hypothesis

Elie Wollman and Francois Jacob (1950s)

The donor (Hfr) strain had the following g ( ) g genetic composition
thr+ : Able to synthesize the essential amino acid threonine leu+ : Able to synthesize the essential amino acid leucine azis : Sensitive to killing by azide (a toxic chemical) tons : Sensitive to infection by T1 (a bacterial virus) lac+ : Able to metabolize lactose and use it for growth gal+ : Able to metabolize galactose and use it for growth strs : Sensitive to killing by streptomycin ( antibiotic) g y p y (an )

The recipient (F) strain had the opposite genotype

thr leu azir tonr lac gal strr r = resistant

Elie Wollman and Francois Jacob (1950s) Experimental design

Mix together large numbers of Hfr and F- cells After different periods of time, interrupt conjugation in a sample of cells using p g a blender
Interrupted mating

Elie Wollman a d Francois Jacob ( 950s) e o a and a co s (1950s) Experimental design

Transfer these thr+ leu+ colonies to a variety of selective medias
Each selective medium tests for the p presence of a p particular transferred allele
Media containing azide selects for azir Media containing T1 phage selects for tonr Media containing lactose as the sole sugar selects for lac+ M di containing galactose as the sole Media i i l h l sugar selects for gal+

The Data
Minutes that Bacterial Cells were Allowed to Mate Before Blender Treatment 5 10 15 20 25 30 40 50 60

There were no surviving colonies after 5 minutes of mating

Percent of Surviving Bacterial Colonies with the Following Genotypes thr+ leu+ azis tons lac+ gal+ 100 100 100 100 100 100 100 100 12 70 88 92 90 90 91 91 3 31 71 80 75 75 78 78 0 0 12 28 36 38 42 42 0 0 0 0.6 5 20 27 27

After 10 minutes, , the thr+ leu+ genotype was obtained The azis gene is then transferred It is followed by the tons gene The lac+ gene enters between 15 and 20 minutes The gal+ gene enters between 20 and 25 minutes

How to count azis clones


Plate without azide Plate with azide

The E. coli Chromosome

Arbitrarily assigned the starting point

Units are minutes Refer to the relative time it takes for genes to first enter an F recipient during a conjugation experiment

the transfer of DNA from one bacterium to another via a bacteriophage Ab t i h bacteriophage iis a virus th t specifically i that ifi ll attacks bacterial cells
It is composed of genetic material surrounded by a protein coat It can undergo two types of life cycles

Lytic Lysogenic

Structure of T4 bacteriophages
Contains the genetic material

Used for attachment to the bacterial surface

It will undergo g the lytic cycle

Prophage can exist in a dormant state for a long time

Virulent phages only undergo a lytic cycle d l ti l

Temperate phages can follow both cycles f ll b th l

phe trp met+ his+

phe+ trp+ met his

Nutrient agar plates lacking the four amino acids Genotypes of surviving bacteria must be phe+ trp+ met+ his+
~ 1 cell in 100,000 was observed to grow

Therefore, genetic material had been transferred between the two strains

However, L d b Lederberg and Zinder obtained novel results when d Zi d bt i d l lt repeating the experiment using the U-tube apparatus (1952)

LA-22 phe trp met+ his+

LA-2 phe+ trp+ met his

Nutrient agar plates lacking the four amino acids

Genotypes of surviving bacteria must be phe+ trp+ met+ his+

No colonies


Process of Transduction The bacterial chromosome is frequently fragmented during a lytic f tl f t dd i l ti infections
A bacterial DNA fragment is sometimes mistakenly packaged into a viral capsid
This DNA can be transmitted to and incorporated into another bacterium i di h b i Generalized transduction Any piece of chromosomal DNA can be incorporated into the phage

Animation h //hi h d hill / i /00 283 12 / d i 0/i d h l

Mapping genes using cotransduction

Cotransduction refers to the packaging and transfer of two closely-linked genes ft l l li k d
It is used to determine the order and distance between genes that lie fairly close together

There is a maximum size to the DNA that can be p packaged by bacteriophages during transduction g y p g g
P1 can pack up to 2-2.5% of the E. coli chromosome P22 can pack up to 1% of the S. typhimurium chromosome

Select for the transduction of one gene M it whether second gene i also t Monitor h th d is l transferred f d Compute cotransduction frequency
= (freq 2nd gene / freq 1st gene) Mathematically determine distance in minutes

Historically relies upon data from both conjugation and transduction j g Conjugation determines relative order and distance between genes di t b t Transduction provides accurate mapping p pp g for genes fairly close together

Transformation is the process by which a bacterium will take up extracellular DNA There are two types
Natural transformation
DNA uptake occurs without outside help

Artificial transformation
DNA uptake occurs with the help of special techniques

Process of natural transformation

Bacterial cells able to take up DNA are termed competent cells competent cells
Numerous gram-negative and grampositive species Carry genes encoding competence factors factors
Facilitate DNA binding to cell surface, uptake into cytoplasm, and integration Expression of these genes is influenced by temperature, ionic conditions, and nutrient availability


6.2 INTRAGENIC MAPPING Intergenic vs intragenic mapping

A plaque is a clear area on an otherwise opaque bacterial lawn on the agar surface of g a petri dish It is caused by the lysis of bacterial cells as a result of the growth and th d reproduction of phages

T4 plaque assay l

T4 phage
Mutations in viral genes can alter plaque morphology
e.g., T4 Rapid lysis mutants destroy cells quickly and form large plaques e.g., Some mutants cannot form plaques

Complementation Tests
Multiple T4 bacteriophage mutants unable to form plaques are isolated E coli is coinfected with two such mutants Plaque formation indicates complementation
Mutations are in different genes

Bacteriophages can be used to map the locations of different mutations within a gene Intragenic mapping or fine structure mapping
Two noncomplementing strains can p p g produce p q plaques at an extremely low rate Distance between mutations affects frequency of ff t f f recombinants

Complementation Tests
E. li E coli K12S Gene A mutation Gene A, B mutation



AB+ Genotype ?

A+ B-

Figure 6.18 describes the general strategy for intragenic mapping of rII phage mutations


r103 r104

Take some of the phage preparation, dilute it greatly 8 (10-8) and infect E. coli B

Take some of the phage preparation, dilute it somewhat 6 (10-6) and infect E. coli K12()

Both rII mutants and wild-type phages can infect this strain

Total number of phages

rII mutants cannot infect this strain

Number of wild-type phages produced by intragenic recombination

66 plaques

11 plaques


Frequency of recombinants
total number of phages 66 X 108 = 6.6 X 109 11 X 106 total number of wild-type phages wildtotal number of recombinants 2X [wild-type plaques obtained in E. coli k12(l)] Frequency of recombinants =
2 (11 X 106) = 3.3 X 103 = 0.0033 6.6 X 109


Genotype ?
Further apart allows more chances for enzyme to bind and function

Deletion Mapping
Deletion mapping can be used to localize many p g gene mutations to specific regions of a g
Involves coinfection of mutant and a deletion strain of the virus Quicker than mapping using large numbers of paired coinfections i f ti

Link to our life

It's not just for Ph.D.s anymore: Amateurs (biohackers) are attempting genetic engineering at home
.In her San Francisco dining room lab, for example, 31-year-old computer programmer Meredith L. Patterson is trying to develop genetically altered yogurt bacteria that will glow green to signal the presence of melamine, the chemical that turned Chinese-made baby formula and pet food deadly. "People can really work on projects for the good of humanity while learning about something they want to learn about in the process," she said.

Cross discipline Heat-Seeking Bacteria Could Hold Key to Better Cellulosic Eth K t B tt C ll l i Ethanol l
We have the organism people have dreamt of -- it eats nearly anything and it makes ethanol really quickly," said Hamish Curran, the company's CEO, showing off TMO's secret weapon several bubbling vats of bacteria in its group laboratories during a recent visit by reporters.

Extension reading
Microbial Genes in the Human Genome: Lateral Transfer or Gene Loss? S. L. Salzberg, O. White, J. Peterson, and J. A. Eisen (8 June 2001) Science 292 (5523), 1903. [DOI: 10.1126/science.1061036]

Widespread Lateral Gene Transfer from Intracellular Bacteria to Multicellular Eukaryotes J. C. D. Hotopp, M E Clark, D. C S G Oliveira, J M F t P. Fischer, J C D H t M. E. Cl k D C. S. G. Oli i J. M. Foster, P Fi h M. C. M. Torres, J. D. Giebel, N. Kumar, N. Ishmael, S. Wang, J. Ingram, R. V. Nene, J. Shepard, J. Tomkins, S. Richards, D. J. Spiro, E. Ghedin, B. E. Slatko, H. Tettelin, and J. H. Werren (21 September 2007) Science 317 (5845) 1753 1756 [DOI 10 1126/ i S i (5845), 1753-1756. [DOI: 10.1126/science.1142490] 1142490]