A mating dance

between cells connected

by conjugation pili.
Microbial
Plasmida circular
snippet of DNA carrying
genetic treasures.
Genetics
Aurora Sinai patient has 'nightmare
bacteria' infection
July 15, 2013
CRE superbug from central line infections is a growing health
problem in hospitals
How do genes, such as those responsible for drug
resistance, arise and/or be transferred between bacterial
species?
 Basic Overview

The genome contains all of the information needed

for the growth and development of an organism
Gene expression refers to the use of DNA sequences
to synthesis RNA and proteins
Review of the Central Dogma of Molecular Biology
 Levels of Genetic Organization

Genome
Chromosome(s) +
Extrachromosomal DNA (ie,
mitochondrial, plasmids) Gene
 Prokaryotic Genomes

Single, circular loop

DNA in nucleoid (cytoplasm)
Usually haploid
Plasmids
Relatively small genome

Chromosome
Plasmids
 Eukaryotic Microbial Genomes

Eukaryotic chromosomes
Multiple, linear
Nucleus Chromosomes
Nucleus
Haploid, diploid or Plasmid (in some
fungi and Nucleolus
polyploid protozoa)
Mitochondrion
Mitochondrial DNA,
chloroplast DNA, plasmids
Large, complex genome Extrachromosomal
DNA

Chloroplast
 DNA Packaging of Bacterial Genomes
No histone proteins (as in
eukaryotes)
The nucleoid forms about
50 loops or domains
Within each domain, the
DNA is supercoiled by DNA-
binding proteins called
nucleoid associated
proteins
 Review of the DNA Packaging of Eukaryotic
Genomes
DNA
Histones and
Condensed metaphase DNA
chromatin chromosome double helix
Supercoiling
of chromatin
DNA Nucleosome
Histone
Supercoiled
condensed Condensed
chromatin nucleosomes

Chromatin
Loosely Uncondensed
condensed chromatin fiber
chromosome
 Bacterial vs Eukaryotic Genome Organization
Bacterial genome Human genome
Packed with genes that ~95% contains noncoding
encode proteins, no introns sequences
Coding genes often occur Coding genes are usually
together in operons single and are often
mRNA is polycistronic; a interrupted by noncoding
transcript encodes >1 introns
proteins mRNA is monocistronic
Processing (poly A tail and 5
cap)
Eukaryotic Genome Organization
Prokaryotic Genome Organization
 Gene Expression in Bacteria

Since bacteria have no nucleus, transcription and

translation can occur in the same location, the cytoplasm,
simultaneously
 Review of Transcription/Translation
Transcription RNA polymerase

DNA
mRNA
Start of
translation
tRNA 50s Ribosomal subunit

30s Ribosomal subunit uses sigma () factor which is a subunit

loosely attached to RNA polymerase that
recognizes the promoter
 Review of Transcription/Translation
DNA

Growing
polypeptides
1

3 Polyribosomal
complex
4
Start
7
5 6
 Alternative sigma factors
Alternative sigma factors recognize different sets of
promoters to control expression of specific groups of
genes
 Prokaryotic Translation Initiation
Initiator tRNA
carrying N-formyl-
methionine
3
1 3 UAC
AUG
2

1 2
mRNA
2 Initiation transcript
1 3 factors start codon
30s ribosomal +GTP
subunit
Translation Initiation

50s ribosomal
subunit
UAC
AUG

mRNA
transcript
 Translation 7 Thr

Elongation
fMet Phe

U G C
U A C E
A A A
A U G U U U A C G G C A C A A G C U G G G
5 3
P A
Peptide bond
formed by 8 fMet Phe Thr

ribozyme
E

5
A A A U G C
A U G U U U A C G G C A C A A G C U G G G
3
Movement of
P A
ribosome
toward 3 end
Growing fMet Phe Thr
Ala Ala
(showing
polypeptide Gln
movement after 2
C G U
E
C G A more cycles
G U U
A U G U U U A C G G C A C A A G C U G G G
5 3
P A
 Review of Translation
DNA triplets

Template
strand tRNAs use their
anticodon to interpret
mRNA the mRNA codons and
codons bring in the amino
UAC GAC UGA UGC
tRNA acids
anticodons
F-Methionine

Threonine
Leucine

Protein
Threonine Same amino acid has a
(amino different codon and
acid specified) anticodon
 Prokaryotic vs Eukaryotic Translation
Both use AUG as start codon but eukaryotes initiator tRNA encodes
unmodified methionine
Eukaryotic ribosomes are 80s (prokaryotes are 70s)
Bacteria often respond to environmental change
by regulating transcription

A cell can regulate the production of enzymes by

feedback inhibition or gene regulation
Gene expression in bacteria is controlled by the operon
model
 Operon
A cluster of functionally related genes can be under
coordinated control by a single on-off switch

regulatory switch

can be switched off by a protein repressor

 Repressor blocks expression of an Operon
Encodes the repressor binds here and
repressor protein blocks RNA polymerase

The repressor can be in an active or inactive

form, depending on the presence of other
molecules
 Components of an Operon
Promoter: RNA polymerase binding site
Repressor: regulatory proteins that binds DNA (operator)
Operator (controlling site): DNA region that is downstream of
promoter and to which the repressor binds
Coding sequences: also known as structural genes, which
specify the expression of proteins involved in a specific
metabolic pathway
 Repressor- a shape shifter!
Each operon is controlled by its own repressor protein
Repressor proteins are allosteric
Allosteric proteins are proteins that change their shape when
bound to a specific molecule called an allosteric effector
This shape change alters ability to bind DNA
 Inducible vs Repressible Operons

Inducible Repressible
Not usually Transcribed continually
transcribed and until deactivated by a
must be activated repressor/corepressor
by inducer Typically regulate
Commonly regulate anabolic pathways
catabolic pathways
Inducible Operon
 Lac Operon from E. coli
Normally E. coli uses glucose but if glucose is unavailable,
lactose can be potentially used as an energy source if the cell
makes enzymes to metabolize lactose
Lac Operon: Contains genes for enzymes that metabolize
lactose
1. Permease (lacY)
Allows lactose to enter bacterial cells
2. Beta-galactosidase (lacZ)
Breaks down lactose to glucose and galactose
3. Transacetylase (lacA)
Unknown function but assists lactose breakdown
 Lac Operon from E. coli

Lac Operon is inducible

o No lactose: repressor prevents transcription
o Lactose present: some converted to allolactose and
allolactose acts as an inducer
o Only occurs when glucose is unavailable
 Lac Operon from E. coli
No lactose in the cell
off

lacZ lacY lacA

(-galactosidase)(permease) (transacetylase)
DNA
Repressor bound Terminator
to operator
RNA polymerase
bound to promoter
 Lac Operon from E. coli
Lactose present in the cell; glucose not available On
Some lactose is converted to allolactose.
lacZ lacY lacA
(-galactosidase)(permease) (transacetylase)

RNA polymerase
Non-functional Transcription
Allolactose
repressor

Translation
 Question
Which line (A, B, or C) represents bacterial growth in
media containing lactose alone?

C
A

B
Repressible Operon
 Arginine Operon
If amino acids are present in the growth medium, E. coli
will import amino acids before it makes them
By default, the arg operon is on and the genes for arginine
synthesis are transcribed
When arginine is present, it binds to the arg repressor
protein, which causes it to change its shape, and thus turn
off the operon
The repressor is active only in the presence of its
corepressor, arginine in this case
 Arginine Operon On
RNA polymerase

Repressor is inactive mRNA

(wrong shape to attach
to operator)
Rep

Arginine is immediately
used in metabolism
 Arginine Operon Off
RNA polymerase

Arginine
Repressor is active accumulates
(correct shape
achieved)
RNA polymerase
 Eukaryotic Gene Regulation

Eukaryotic regulation more complicated

Variety of approaches
o Modification of chromosome structure
o Regulation of initiation of transcription
o Altering pre-mRNA processing and modification
o RNA interference (RNAi)
 Genomes Evolve and Change
Mutation Horizontal gene transfer
 Natural Selection
In a population of organisms that experience a change
in their environment, organisms that are more fit to
thrive in a new environment will predominate a
population
Mutation and horizontal gene transfer provide
diversity. The fittest organisms for an environment are
then selected by natural selection
 Genetic Change
Change in organisms DNA alters genotype
o Sequence of nucleotides in DNA
o Bacteria are haploid, so only one copy, no backup

May change observable characteristics, or phenotype

Geneticists compare mutants

to wild type
o Typical phenotype of strains
isolated from nature
 Review of DNA Replication
DNA is synthesized by DNA polymerase with the help of accessory
proteins
o Creates complementary strand to a template strand
Initiates at an origin of replication (ori)
Bacteria have a single origin of replication (eukaryotes have
multiple origins )
 Review of DNA Replication Original

As the termination site is replicated, topoisomerase strand

separates to the 2 loops of DNA

DNA Replication

New
strand

Origin of
replication
Original
strand

Replication
forks
Site where
replication
ends New
topoisomerase strand

Replication of Bidirectional replication

chromosomal DNA creates The replication forks DNA replication is
starts at the origin of two advancing forks ultimately meet at a semiconservative,
replication and then where DNA synthesis is terminating site.
(a) meaning each of the two
proceeds in both occurring.
molecules created contains one
directions.
of the original strands paired
with a newly
synthesized strand.
 Spontaneous Mutation
Arise during normal DNA replication
Occur randomly at infrequent but characteristic rates
Mutation rate: probability of mutation each cell division
oTypically between 104 and 1012 for a given gene
Mutations passed to progeny
Occasionally change back to original state: reversion
Large populations contain mutants (e.g., cells in colony)
 Question
Why would a frameshift mutation in the gene for
topoisomerase be lethal for E. coli but not for a human
cell that also uses topoisomerase during DNA
replication?
 Mutation
Because bacteria are unicellular, mutations, if not
repaired, are always passed onto their progeny
Mutations may be neutral, beneficial, or harmful

1. Spontaneous Mutations
o Occur randomly due to errors in DNA replication

2. Induced Mutations
o Caused by exposure to mutagens (physical or chemical)
 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction

Base substitution most 5' 3'

common 3' 5'

DNA strand separation

5' 3'
DNA replication, an
Incorrect nucleotide DNA replication incorrect
3' 5'
nucleotide is
incorporated during incorporated
5' 3' 5' 3'
DNA synthesis Base substitution
3' 5' 3' 5'
Wild type
DNA strand separation
Point mutation is change DNA 5' 3'

replication,
of a single base pair generating 3' 5'
DNA
a mutation replication
5' 3' 5' 3'

3' 5' 3' 5'

Mutant Wild type

 Consequences of Base Substitution

Silent mutation: wild-type amino acid (redundancy of genetic

code)

Missense mutation: different amino acid

Resulting protein may only partially function leaky

Nonsense mutation:
Specifies stop codon
Resulting protein is shorter and typically non-functional
A mutation that inactivates a gene is termed a null or
knockout mutation
Consequences of Base Substitution
Consequences of Base Substitution
Consequences of Base Substitution
 Genetic Code has Redundancy,
degenerate
Second Base Position
U C A
G
UUU
UUC }Phenylalanine UCU
UCC
UAU
UAC
} Tyrosine UGU
UGC } Cysteine
U
C
U
Serine
UUA
UUG } Leucine UCA
UCG
UAA
UAG
} STOP** UGA
UGG
STOP*
Tryptophan
*
A
G

CUU
CUC
CCU
CCC
CAU
CAC
} Histidine CGU
CGC
U
C
C
Leucine Proline Arginine

Third Base Position

} Glutamine CGA
First Base Position

CUA CCA CAA A

CUG CCG CAG CGG G

AUU
AUC Isoleucine
ACU
ACC
AAU
AAC
} Asparagine } Serine
AGU
AGC
U
C
A
Threonine
AUA
AUG* STARTMethionine
AC A
ACG
AAA
AAG
} Lysine AGA
AGG
} Arginine
A
G

GGU
GUU
GUC
GCU
GCC
GAU
GAC
} Aspartic acid
GGC
U
C
G
Valine Alanine Glycine
GUA
GUG
GC A
GCG
GAC
GAG
} Glutamic acid
GGA
GGG
A
G

*This codon initiates translation.

**For these codons, which give the orders to stop translation, there are no corresponding tRNAs with amino acids.
 Consequences of Deletion or Insertion of
Nucleotides
Impact depends on number of nucleotides
Three pairs changes one codon
One amino acid more or less
Impact depends on location within protein

One or two pairs yields frameshift mutation

Different set of codons translated
Often results in premature a stop codon
Shortened, nonfunctional protein
Null or Knockout mutation
Consequences of Deletion or Insertion of Nucleotides
Consequences of Deletion or Insertion of Nucleotides
Consequences of Various
Types of Mutations
 Question
Which type of mutation is generally the most
serious?

A. Missense
B. Insertion of 3 nucleotides
C. Silent
D. Nonsense
 Induced Mutations

Result from outside influence

Agent that induces change is mutagen
Geneticists may use mutagens to increase mutation
rate
Two general types: chemical and physical
 Physical Mutagen: Radiation
(a) Ionizing radiation (X-rays)
Single and double-stranded
breaks in the DNA
Double stranded breaks
often produce lethal
deletions
(b) Nonionizing radiation (UV)
Thymine dimers
Covalent bonds between
adjacent thymines
 Chemical Mutagens- Alkylating Agents
Add methyl or ethyl groups to nucleobases
Change base-pairing properties
Increase chance of incorrect nucleotide incorporation
Hydrogen bonds formed
With complementary bases
CH3 Added
alkyl group
O O
N H
N
N N
H H H H
N N N N
N Alkylating agent N
deoxyribose H deoxyribose H

Guanine Methylguanine
(pairs with C) (sometimes pairs with T)
 Chemical Mutagens- Alkylating Agents

Wild type Mutant

3' DNA 3' DNA 5' 3'
5' 3' 5' 5'
treatment AG *T replication AG*T replication AAT
AGT TTA
T CA T CA T TA
3' 3' 5' 3' 5' 3' 5'
5'
Thymine (formerly paired
Guanine (G) is with G*) now serves as
converted template and pairs with
to adenine (A)
methylguanine Methylguanine of template
(G*) strand pairs with thymine (T)
(b) instead of cytosine (C)
 Chemical Mutagens- Base Analogs
Normal
Analog
Resemble nucleotide bases
nucleobase
O O

Have different hydrogen- H3C 6 N H

5 1 Replaced by
Br 6 N H
5 1
43 2 43 2
bonding properties N O N O
H H
Can be mistakenly Thymine
(pairs with A)
5-bromouracil
(pairs with A or C)
incorporated by DNA H H
N N
polymerase N 7 5 6 1N
7 5 6 1N Replaced by
8
9 4 3 2 H
8 4 2 N N N
9 3 H H
N N H
H
Adenine 2-amino purine
(pairs with T) (pairs with T or C)
 Chemical Mutagens- Intercalating Agents
Flat molecules that intercalate
(insert) between adjacent base
pairs in DNA strand
Pushes nucleotides apart,
produces space
Cause the addition or
deletion of a single base
during replication
frameshift mutations
 DNA Repair Mechanisms
Many mechanisms
Some examples:
Prevention: DNA polymerase
proofreading activity

Mismatch repair:
o Fixes errors missed by the
proofreading of DNA
polymerase
o Mismatch repair enzymes
recognize unmethylated DNA
 Horizontal Gene Transfer
Exchange of genes between bacteria of
the same generation
Donor cell contributes part of its
genome to recipient cell, which may be
of a different species and/or genera
Rare event
Genetic exchange between bacteria:
1. Conjugation
2. Transformation
3. Transduction
 Maintenance of Transferred Genes in a
Population

DNA must be part of a replicon to be maintained in a

population
Plasmids, chromosomes
DNA fragments added to chromosome via homologous
recombination
Only if sequence similar to region of recipients genome
Non integrated DNA fragment
Integrated DNA fragment
 Transformation
Naked DNA in solution is taken up by recipient cells

Inhibited by DNAse

Frederick Griffith demonstrated genetic transformation

using mice and Streptococcus pneumoniae
Transformation
Transformation
 Question
DNAse is an enzyme that degrades DNA. If DNAse had been added to
Griffiths last experiment in which he injected a mouse with mix of heat
killed encapsulated cells and live non-encapsulated cells, then how
would the result have been different?

A.The mouse would not have died

B.The bacteria isolated from the mouse would have been a mix of live
non-encapsulated cell and live encapsulated cells
C.The bacteria isolated from the mouse would have been live non-
encapsulated cells
D. A and C
 Competence and the Process of
Transformation

The recipient cell must be competent

Competence: a specific physiological state that allows the
cell to take up DNA
Only a few genera of bacteria are naturally competent
Transformasomes capture naked free-floating DNA and
transports one strand into the cell while degrading the
other strand
 Transduction

The process by which bacterial viruses, called

bacteriophages (phages), transfer bacterial genes from
a donor to a recipient

2 Types :
1. Generalized transduction which involves a lytic
bacteriophage
2. Specialized transduction which involves a temperate
bactiophage
Replication Cycle of Lytic vs Temperate Bacteriophages

Induction
 Generalized Phage Phage attaches to specific receptor of
Transduction bacterial cell (donor cell)
Step 1
Phage DNA enters cell.

Phage enzymes cut the bacterial DNA into

small pieces
Transducing particle Phage nucleic acid is replicated and
carries bacterial DNA phage proteins synthesized
instead of phage
DNA During assembly of viral particle, bacterial
DNA can mistakenly enter a protein coat.
 Generalized
A transducing particle attaches to
Transduction a specific receptor on host cell
Step 2 (recipient cell)

Bacterial DNA is injected into

the cell.

The injected bacterial DNA

integrates into the chromosome
by homologous recombination

Bacteria multiply with new genetic

material. Replaced host DNA is
degraded.
 Specialized Transduction

A temperate phage injects its

DNA into a bacterial cell

The phage DNA integrates into the host cell DNA

at a specific integration site to become a
prophage

When the prophage is excised from the bacterial

chromosome, a mistake is made; some bacterial DNA
flanking the phage DNA is taken and a piece of the
phage DNA is left behind
 Specialized Transduction

Replication and assembly produces defective

phage particles that carry certain bacterial DNA in
place of some phage DNA

The DNA of the defective phage is injected into

the new host but cannot cause a productive
infection

The bacterial DNA integrates into the host genome via

homologous recombination; it can now be replicated
along with the rest of the host DNA
Integrated DNA from donor bacterial cell
 Bacterial Conjugation
Genetic transfer via direct contact
Gram positive and gram negative bacteria can both transfer DNA via
conjugation, but the process is different in the 2 groups

We will consider conjugation only in the Gram negative bacteria

Sex pilus
 F plasmid Origin of Conjugation pilus
transfer

Bacterial Conjugation
Involving Transfer of a F cell F cell
free F Plasmid

F cell F cell
 Conjugation involving Chromosome Transfer
Cells in which the F plasmid has integrated into the
chromosome by homologous recombination are called
Hfr cells (high frequency of recombination)

In E.coli:
Time to transfer the free F plasmid= 5 minutes
Time to transfer the entire E. coli chromosome=
100 minutes

Sex pilus cannot be maintained for long enough to

transfer an entire chromosome
 Donor chromosome
Conjugation involving Pilus
Chromosome Transfer
F+ cell
Integrated F plasmid
Hfr cell
Pilus F recipient
F+ cell (Hfr)

F plasmid Donor DNA

Origin of transfe

Incomplete F plasmid;
cell remains F

Recombinant cell (still F)

 Question
If an HFR strain conjugates with an F- strain, what is the
result?
A. Both strains will be F+
B. HFR strain remains the same but the F-
becomes a recombinant
C. Both strains remain the same
D. The HFR strain becomes F- but the other
strain becomes F+
E. Both strains are HFR
 Question
You are performing a conjugation experiment with the following strains:
Strain A: erythromycin sensitive, ampicillin resistant
Strain B: erythromycin resistant, ampicillin sensitive
Resistance to erythromycin is carried on the chromosome, ampicillin
resistance is carried on a plasmid. After incubating the strains together
(giving ample time for conjugation to occur), you plate the conjugation
mixture on a variety of media. Which of the following plates will select
for growth of conjugants?

A. LB agar
B. LB agar + ampicillin
C. LB agar + erythromycin
D. LB agar + erythromycin + ampicillin
 Mobile Genetic Elements
Carry genes from one DNA molecule to
another
oPhage DNA
oPlasmids
oTransposons
 Plasmids

Have origins of replication and therefore can replicate

independently from the chromosome
Low-copy-number: 1-3 per cell
High-copy number: many, up to 500
Are most frequently transferred to other cells by
conjugation
 Plasmids

Code for non-essential functions

but may confer beneficial traits

R plasmids: plasmids that carry

antibiotic resistance genes (and
often pilin synthesis genes, ie F
factor)
 Transposition (1)

Transposable elements provide

mechanism for moving DNA
Can move into other replicons in
same cell
Integrate via non-homologous
recombination (2) (3) (4)
 Transposable Elements

Integrate into their new location through non-homologous

recombination, a process that does NOT require extensive
areas of homology between the transposan and its
destination site but does require specialized recombination
enzymes encoded by the transposon
 Insertion Sequences are the simplest
Transposable Elements
Insertion sequence

Mobile element

Transposase gene
Inverted repeat Inverted repeat

5 3 5 3
T C G A T G CA T C G A
A G C T AC ....GT A G C T
3 5 3 5
 Transposan
Are transposable elements that contain the gene for
transposase as well as other genes that are not required
for transposition (ie antibiotic resistance or toxin genes)
Composite transposon

Mobile element

Insertion Antibiotic-resistance
Insertion
sequence gene
sequence
 Transfer of Antibiotic
Resistance Genes from
normal microbiota to a
pathogen
Review of Horizontal Gene Transfer
Review of Horizontal Gene Transfer