Bidirectional DNA replication:

http://highered.mheducation.com/sites/0072556781/student_view0/chapter11/animation_quiz_1_.htm
l
Rolling Circle DNA replication:
http://highered.mheducation.com/sites/0072556781/student_view0/chapter13/animation_quiz_6.html

PSU BI 341 CHAPTER 7

THE GENETICS OF BACTERIA AND
THEIR VIRUSES
Kim H Brown, PHD

STAPHYLOCOCCUS AUREUS,
COMMONLY CARRIED ON
HUMAN SKIN AND NASAL
PASSAGES.
 Objectives
1) The utility of bacteria as a model system
a) Mobility of bacterial DNA: Conjugative plasmids,
transposons, integrons
i) Hfr and Bacterial chromosomal mapping
(1)The power of mutational analysis
(2)Unidirectional gene transfer
(3)Time of entry mapping
2) Bacteriophages
a) Generalized
b) Specialized
c) Temperate
Bacterial gene transfer and antibiotic
i) Lytic vs. Lysogeny
resistance: http://
www.youtube.com/watch?v=tQZnhjJnD
5E
 Plasmids
• Many DNA sequences in bacteria are mobile and can be
transferred between individuals and among species.
• Plasmids are non-essential circular DNA molecules that replicate
independently of the bacterial chromosome.
• Plasmids often carry antibiotic resistance genes
• Plasmids are used in genetic engineering as gene transfer
vectors

Electron micrograph of a ruptured E.

coli cell, showing released
chromosomal DNA and several
plasmid molecules.

http://highered.mheducation.com/sites/007255
6781/student_view0/chapter14/animation_quiz_
2.html
 Bacterial Transformation
• The process of genetic alteration
by pure DNA is transformation.
• Recipient cells acquire genes
from DNA outside the cell.
• DNA is taken up by the cell and
often recombines with genes on
bacterial chromosome.
• Bacterial transformation showed
that DNA is the genetic material.

Bacterial Transformation:
http://highered.mheducation.com/sites/007255
6781/student_view0/chapter13/animation_quiz_
1.html
 Cotransformation of Linked Genes
• Cotransformation: genes located
close together are often transferred
as a unit to recipient cell.
• Cotransformation of two genes at a
frequency substantially greater than
the product of the single-gene
transformation frequencies implies
that the two genes are close
together in the bacterial
chromosome.
• Genes that are far apart are less
likely to be transferred together
• Cotransformation is used to map
gene order
 Transposable Elements
• Transposable elements are DNA sequences that can
jump from one position to another or from one DNA
molecule to another
• Bacteria contain a wide variety of transposable
elements
• The smallest and simplest are insertion sequences,
or IS elements, which are 1–3 kb in length and encode
the transposase protein required for transposition and
one or more additional proteins that regulate the rate
of transposition

Simple Transposition: http://

highered.mcgraw-hill.com/sites/0072995246/student_view0/chapter23/simple_transpo
sition.html
 Transposable Elements
• Other transposable elements in bacteria contain one or more genes
unrelated to transposition that can be mobilized along with the
transposable element; this type of element is called a transposon
• Transposons can insert into plasmids that can be transferred to
recipient cells by conjugation
• Transposable elements are flanked by inverted repeats and often
contain multiple antibiotic resistance genes

What causes antibiotic

resistance?
http://ed.ted.com/lessons/how-a
ntibiotics-become-resistant-ove
r-time-kevin-wu
 Integrons: site specific transposable
elements
• Integron is a DNA element that
encodes a site-specific
recombinase and a recognition
region that allows other
sequences with similar
recognition regions to be
incorporated into the integron
by recombination.

• The elements that integrons acquire are known as cassettes

• Integrons may acquire multiple-antibiotic-resistance
cassettes
• Bacteria with resistance to multiple antibiotics are an
increasing problem in public health
 F factor and Conjugation
• F (fertility) factor is a conjugative plasmid transferred from
cell to cell by conjugation
• F factor is an episome–a genetic element that can insert into
chromosome or replicate as circular plasmid
• The F plasmid is a low-copy-number plasmid ~100 kb in length
and is present in 1–2 copies per cell
• It replicates once per cell cycle and segregates to both
daughter cells in cell division

PILUS CONNECTING TWO CELLS OF E. COLI,

ALLOWING GENE TRANSFER BETWEEN THE
CELLS. THE CELL WITH MULTIPLE
APPENDAGES CONTAINS AN F PLASMID THAT
ENCODES THE ~20 GENES NECESSARY TO
PRODUCE THE PILUS.
 F factor and Conjugation
• Conjugation is a process in which
DNA is transferred from bacterial
donor cell to a recipient cell by cell-to-
cell contact
• Cells that contain the F plasmid are
donors and are designated the F+
• Cells lacking F are recipients and are
designated the F–
• The transfer is mediated by a tube-
like structure called a pilus, formed
between the cells, through which the
plasmid DNA passes

F Factor transfer
http://highered.mcgraw-hill.com/sites/0072556781/student_view
0/chapter13/animation_quiz_3.html
 Conjugation
• In bacterial mating, conjugation,
DNA transfer is unidirectional
• F factor can integrate into
chromosome via genetic
exchange between IS elements
present in F and homologous
copy located anywhere in
bacterial chromosome
• Cells with the F plasmid integrated
into the bacterial chromosome are
known as Hfr cells
• Hfr: High Frequency of
Recombination
 Hfr
• In an Hfr cell the bacterial
chromosome remains circular,
though enlarged ~ 2 percent by the
integrated F-factor DNA
• When an Hfr cell undergoes
conjugation, the process of
transfer of the F factor is initiated in
the same manner as in an F+ cell
• However, because the F factor is
part of the bacterial chromosome,
transfer from an Hfr cell also
includes DNA from the
chromosome
 Hfr and Conjugation
• Transfer begins within an
integrated F factor and proceeds in
one direction
• A part of F is the first DNA
transferred, chromosomal genes
are transferred next, and the
remaining part of F is the last
• The conjugating cells usually break
apart long before the entire
bacterial chromosome is
transferred, and the final segment
of F is almost never transferred
• The recipient cell remains F–
 Chromosome Mapping
• It takes 100 minutes for an entire bacterial
chromosome to be transferred and about 2
minutes for the transfer of F
• The difference reflects the relative sizes of F and
the chromosome (100 kb versus 4600 kb)
• Regions in the transferred DNA may incorporate
into the recipient chromosome and replace
homologous regions
• This results in recombinant F– cells containing
one or more genes from the Hfr donor cell

Conjugation: Transfer of Chromosomal DNA

http://highered.mheducation.com/sites/0072556781/st
udent_view0/chapter13/animation_quiz_4.html
 Bacterial Genetics: allowing
identification of gene transfers
• Three principal types of bacterial mutants use in bacterial
genetics:
1. Antibiotic-resistant mutants are able to grow in the
presence of an antibiotic.

Example
Str-s strain is streptomycin
sensitive, so cannot grow on
media containing
streptomycin
Str-r is a mutant strain which
is streptomycin resistant
 Bacterial Genetics: allowing
identification of gene transfers
• Three principal types of bacterial mutants use in bacterial
genetics:
2. Nutritional mutants are unable to synthesize an essential
nutrient and thus cannot grow unless the required nutrient
is supplied in the medium. Such a mutant bacterium is
said to be an auxotroph. Prototrophs are able to
synthesize their own nutrients in minimal media.
3. Carbon-source mutants cannot utilize particular
substances as sources of energy or carbon atoms.
Production of prototrophs as a result of
recombination between auxotrophs
Recall the mechanism of gene transfer in Hfr
cells
Gene order is indicated by recombination
frequency
 Chromosome “time of entry” Mapping &
Interrupted Mating
1. In this example, the str-r gene
a counterselected marker,
e.g is only in the F- strain and
allow for selection of this F-
recipient strain.
2. The leu gene is the
transferred gene, or the
selected marker.
3. The mating is set up as such:
Hfr leu+ X F- leu-str-r
Important Notes:
1)Only the F- str-r will grow in the
The mating is interrupted, and
presence of streptomycin
bacteria is grown in the 2)Only those F- recombinants that
presence of strep, and have gained a leu gene from the
absence of leucine Hfr donor will grow without leucine
 Chromosome “time of entry” Mapping &
Interrupted Mating
Hfr leu+ X F- leu-str-r
4.Thus, leu is 6 map units (6
minutes) from origin of transfer
5.In general the matings are
set up similar to a test cross:

Hfr a+b+c+d+e+str-s X
F- a-b-c-d-e-str-r
 Chromosome “time of entry” Mapping &
Interrupted mating
1. Genes in the bacterial
chromosome can be
mapped by Hfr x F–
mating
2. DNA transfer can be
interrupted by “violent
agitation”.
3. The number of
recombinants genes
increases with time of
contact between cells
4. Resulting map is based
on order of transfer,
with 1 map unit = 1
minute transfer time
 The result of time of entry Mapping

Circular genetic map of E. coli shows map

distances of genes in minutes
Circular genetic map of E. coli
 Viruses
Bacteriophage: a virus that infects and replicates within a
bacterium. The term is derived from "bacteria" and the Greek:
φαγεῖν, "to devour".
http://www.npr.org/blogs/krulwich/2011/06/01/11407502
9/flu-attack-how-a-virus-invades-your-body
 Transduction
• In the process of transduction,
bacterial DNA is transferred from
one bacterial cell to another by a
phage
• A generalized transducing
phage transfers DNA derived
from any part of the bacterial
chromosome
• A specialized transducing
phage transfers genes from a
particular region of the bacterial
chromosome.
Generalized Transduction:
http://highered.mheducation.com/sites/0
072556781/student_view0/chapter13/anima
tion_quiz_2.html
 Generalized Transduction: An example
phage P1
• A generalized transducing
phage P1 cuts bacterial
chromosome into pieces and
can package bacterial DNA
into phage particles –
transducing particle
• Transducing particle will insert
‘transduced” bacterial genes
into recipient cell by infection
• Transduced genes may be
inserted into recipient
chromosome by homologous
recombination
 Generalized Transduction
• Typical P1 transducing
particle contains from 100-
115 kb of bacterial DNA
(about 50 genes)
• Probability of
cotransduction of both
markers depends
proximity of genes.The
closer they are, the
greater the frequency of
cotransduction
• Cotransduction provides a
valuable tool for genetic
linkage studies
 Specialized Transduction
• Specialized transducing
phages transduce bacterial
genes at the site of prophage
insertion into the bacterial
chromosome
• Transduction of bacterial genes
occurs by aberrant excision of
viral DNA, which results in the
incorporation of bacterial genes
into phage chromosome

Specialized Transduction:
http://highered.mheducation.com
/sites/0072556781/student_view0
/chapter17/animation_quiz_3.htm
l www.bio.miami.edu
 Temperate Bacteriophages
• Temperate bacteriophages have two
life cycles:
 lytic cycle = infection that results in
production of progeny phage and
bacterial cell lysis
 lysogeny = nonproductive viral
infection results in insertion of viral
DNA into bacterial chromosome
• Viral DNA integration = site-specific
insertion into bacterial chromosome

Lambda Phage Replication Cycle:

http://highered.mheducation.com/sites/0072556781/stu
dent_view0/chapter17/animation_quiz_2.html
facstaff.cbu.edu
 Lytic Cycle
• The reproductive cycle of a phage is called the lytic cycle
• In lytic cycle: Phage DNA enters the cell and replicates
repeatedly Cell ribosomes produce phage proteins
• Phage DNA and proteins assemble into new phage particles
• Bacterium is split open (lysis), releasing phage progeny with
parental genotypes
 Lytic Cycle
• When two phage particles that have different genotypes
infect a single bacterial cell, new genotypes can arise by
genetic recombination
• This process differs from genetic recombination in
eukaryotes:
 the number of participating DNA molecules varies
from one cell to the next
 reciprocal recombinants are not always recovered in
equal frequencies from a single cell
The Seymour Benzer Model
 Benzer experiments:
Fine Structure of the Gene
• The mutation and mapping
studies of rII locus of phage T4
performed by S. Benzer
provided an experimental proof
to important conclusions:
 Genetic exchange can take
place within a gene and
probably between any pair of
adjacent nucleotides
 The unit of mutation is an
individual pair of nucleotides
 Mutations are not produced at
equal frequencies at all sites Array of deletion mutations used to
within a gene divide the rII locus of phage T4.
 Genetic map of part of the rII locus of phage
T4.

Adapted from S. Benzer,

Proc. Natl. Acad. Sci. USA 47(1961): 403-426
 Lysogenic Cycle
• All phage species can undergo a lytic cycle
• Phages capable of only the lytic cycle are called
virulent
• The alternative to the lytic cycle is the lysogenic
cycle: no progeny particles are produced, the
infected bacterium survives, and a phage DNA is
transmitted to each bacterial progeny cell when
the cell divides
• Those phages that are also capable of the
lysogenic cycle are called temperate
 Lysogenic Cycle
• In the lysogenic cycle, a replica of
the infecting phage DNA becomes
integrated into the bacterial
chromosome
• The inserted DNA is called a
prophage, and the surviving
bacterial cell is called a lysogen
• Many bacterial generations, after a
strain has become lysogenic, the
prophage can be activated,
excised from the chromosome,
and the lytic cycle can begin
 Bacteriophage 
• E. coli phage  is a temperate
phage capable of both lytic
and lysogenic, cycles
• The DNA of  is a linear
molecule with cohesive ends
(cos) that pairing yields a
circular molecule
• In lysogen, prophage  is
linearly inserted between the
gal and bio genes in the
bacterial DNA
 Bacteriophage 
• Prophage genetic map is a
permutation of the genetic map
of the phage progeny obtained
from standard phage crosses.
• Upon induction, the prophage 
is usually excised from the
chromosome precisely.
• However, once in every 106 or
107 the excision error leads to
formation of aberrant phage
particles that can carry either the
bio genes (cut at the right) or the
gal genes (cut at the left)