Microbial Genetics

• Lecture 1

Prokaryotic Genetics

Horizontal gene
transfer in bacteria
 Introduction

• Bacteria posses basic three mechanisms of lateral or horizontal

gene transfer. They are,

 
– Transformation- transfer of gene from one bacterium to other without
cell to cell contact.

–  Transduction– transfer of gene from one bacterium to other via phages.

– Conjugation- transfer of gene from one bacterium to other by cell to cell

contact by conjugal pore. Conjugation was first discovered in 1946 by
two renowned scientists of the time  Joshua Lederberg and Edward
Tatum.
Conjugation
Distinguishing characters of conjugation

• DNA transfer requires cell-cell contact.

• DNA transfer occurs via a conjugal pore.

• DNA transfer occurs in one direction - from donor to recipient not vice
versa .

• DNA transfer does not require protein synthesis in donor.

• DNA transfer requires energy in donor cell - primarily ATP.

F+ (donor)

Conjugal bridge

F- (recipient)
 Fertility plasmid
•  The E. coli fertility plasmid is the most extensively studied plasmid. 

• The general features of this plasmid are,

– 1. Large circular plasmid (100 kb)

– 2. Only 60% genes has been mapped.

– 3. 32 kb is organized as a unit to transfer its genome to another bacterium

( transfer region or tra genes)

– 4. Two methods of replication :

a. oriV  as free plasmid
(one copy/ bacterial chromosome)
b. uses bacterial chromosomal origin when integrated
(oriC);oriV is suppressed.
 Mechanism

• Plasmid replication requires a "mating bridge" between the donor

and recipient cells.
•  Before the mating bridge can form:- donor must recognize
recipient cell- donor must make contact with recipient cell
• The conjugative functions of the F plasmid are specified by a
cluster of at least 25 transfer (tra) genes.
• They determine:
– expression of F pili.
– synthesis and transfer of DNA during mating
– interference with the ability of F+
– bacteria to serve as recipients.
• Each F+ bacterium contains F pili
• Binding of F pili to specific outer membrane protein
• Intercellular cytoplasmic bridge formation 
• Transfer of single strand from donor to recipient 
• Transferred strand is converted into double stranded circle
in recipient. 
• The copy is retained in donor The newly synthesized double
stranded DNA is called exogenote and native DNA as
endogenote.
 Domain interacting with outer membrane
protein

3D X ray crystallographic structure of protein pilin

General representation of mechanism of conjugation
The relaxase and the relaxosome
When conjugation is initiated, via a mating signal, a relaxase enzyme (an
endonuclease) creates a nick in one plasmid DNA strand at the origin of transfer, or
oriT.

The relaxase may work alone or in a complex of over a dozen proteins, known
collectively as a relaxosome.

In the F-plasmid system, the relaxase enzyme is called TraI and the relaxosome
consists of TraI, TraD, TraY, TraM, and the Integrated Host Factor, IHF.

The transferred, or T-strand, is unwound from the duplex plasmid and transferred
into the recipient bacterium in a 5'-terminus to 3'-terminus direction.

 
• The remaining strand is replicated, either independent
of conjugative action (vegetative replication, beginning at the oriV) or in
concert with conjugation (conjugative replication similar to the rolling circle
replication of lambda phage).
 Initiates F plasmid
transfer

Helps creating
the ssDNA
conformation

Sterically
prepare the
DNA for other
proteins to
interact in
specific spots.
Conjugation protein assembly
Domains of protein tra I
Bacterial Conjugation:
High Frequency Transfer (Hfr) Cells
 Bacterial Conjugation:
High Frequency Transfer of DNA

Different
Alleles
Bacterial Conjugation:
F ´ Transfer
 Mapping Gene Position in Bacteria:
Interrupted Conjugation Mapping

r
s

Donor Hfr Cell Recipient F- Cell

(wildtype) (mutant)
 r
s

Time Zero
Plate: Selective Meida w/ Streptomycin (no Hfr) and w/o threonine and leucine (no F-
cells that have not received DNA)

Replica Plate: Selective Media for other genes

w/ azide w/ phage w/ lactose w/ galactose

carbon source carbon source
 Mapping Gene Position in Bacteria:
Interrupted Conjugation Mapping

F- leu+, thr+ cells

Replica Plate: Selective Media

w/ azide w/ phage w/ lactose w/ galactose

carbon source carbon source
 Mapping Gene Position in Bacteria:
Interrupted Conjugation Mapping
F- leu+, thr+ cells
s

Replica Plate: Selective Media

w/ azide w/ phage w/ lactose w/ galactose

carbon source carbon source
Mapping Gene Position in Bacteria:
Interrupted Conjugation Mapping

s
Mapping Gene Position in Bacteria:
Map Distance is in Minutes
 Building a Chromosomal Map:
Several Hfr Strains
Hfr1 Hfr5
 Building a Chromosomal Map:
Transfer Alignment

Unaligned Transfers Aligned Transfers

Transformation