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PART

How Genes Travel on Chromosomes CHAPTER


IV

Bacterial Genetics

CHAPTEROUTLINE
13.1 The Enormous Diversity of Bacteria
13.2 Bacterial Genomes
13.3 Bacteria as Experimental Organisms
13.4 Gene Transfer in Bacteria
13.5 Bacterial Genetic Analysis
13.6 A Comprehensive Example: How N. gonorrhoeae Became Resistant to Penicillin

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Hartwell et al., 5th ed., Chapter 13
Bacteria constitute one of the three major
evolutionary lineages

In an average human, there


are 9 times as many
bacterial cells as human
cells.
Most bacteria are in the
intestines, but the skin,
mouth, and respiratory
tract are also homes to
bacteria.
Bacteria aid human health
and some cause disease.

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Hartwell et al., 5th ed., Chapter 13
13.1 The Enormous Diversity of Bacteria

Learning objectives:
List key features of prokaryotic cells.

Discuss how bacterial habitats influence bacterial


metabolisms.

Summarize the properties that make certain bacteria


pathogenic to humans.

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Hartwell et al., 5th ed., Chapter 13
Characteristics of bacteria

Come in a variety of shapes and sizes


Lack a nucleus and membrane bound organelles
Have a single chromosome
Most have a cell wall

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Hartwell et al., 5th ed., Chapter 13
Bacteria have adapted to a range of habitats

Different habitats
On land, in aquatic environments, as parasites or symbionts
inside other life-forms
Some bacteria cause hundreds of animal and plant disease

Most are crucial to maintenance of earth's environment


Release oxygen to atmosphere
Recycle carbon, nitrogen, and other elements
Digest human and other animal waste
Neutralize pesticides and other pollutants
Produce vitamins and other materials essential to humans
and other organisms
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Hartwell et al., 5th ed., Chapter 13
A small fraction of bacteria are pathogens

Pathogen bacterial strain that causes disease


Pathogenic bacteria:
Invade tissues

May produce toxins, proteins that interfere with cell function


or destroy cells

Tetanus toxinresults in paralysis by interfering with


communication between nerves and muscles

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Hartwell et al., 5th ed., Chapter 13
13.2 Bacterial Genomes

Learning objectives:
Describe how genes are organized within a bacterial genome.

Differentiate between a species core genome and its


pangenome.

Discriminate between IS and Tn transposable elements in


bacteria.

Describe how plasmids conferring multidrug resistance to


bacteria may have evolved.

Explain how the study of metagenomics might yield practical


benefits.

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Hartwell et al., 5th ed., Chapter 13
The typical bacterial genome is composed of
one circular chromosome

4 to 5 Mb of DNA in most
commonly studied bacterial
species

DNA molecule condenses by


supercoiling and looping

Each bacterium replicates and


then divides by binary fission
into two daughter cells

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Hartwell et al., 5th ed., Chapter 13
The bacterial chromosome

Usually circular
4-5 Mb = 1.6 mm long = 1000 times longer than the cell
Packaged by supercoiling to fit inside cell.

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Hartwell et al., 5th ed., Chapter 13
Bacterial genomes contain transposons

Insertion sequences (IS) are like eukaryotic transposable


elements:
Inverted repeats at the ends

Encodes transposase

Can disrupt gene function

Can rearrange bacterial genomes by causing deletions or


inversions

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Hartwell et al., 5th ed., Chapter 13
Bacterial genomes contain transposons

Tn elements are composite transposable elements.


Two nearby transposable elements

Flank a gene for resistance to antibiotics or toxic metals.

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Hartwell et al., 5th ed., Chapter 13
Plasmids carry additional DNA

Plasmids
Small circles of double
stranded DNA

Used as cloning vectors

May contain genes that


benefit host bacterium or
contribute to bacterial
pathogenicity

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Hartwell et al., 5th ed., Chapter 13
Plasmids may provide resistance to antibiotics

Resistance plasmids
Contain composite IS/Tn
transposons.

Carry genes that confer


resistance to multiple
antibiotics

Can be easily transferred


from one bacterium to
another in nature.

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Hartwell et al., 5th ed., Chapter 13
Bacteria must be grown and studied in cultures

Bacteria grown as Bacteria grown as colonies


a cell suspension on solid nutrient-agar in a
in liquid media petri dish

Fig. 14.2

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Hartwell et al., 5th ed., Chapter 13
Escherichia coli (E. coli) is
a versatile model organism

E. coli is the most studied and best understood bacterial


species
Normally inhabits intestines of warm-blooded animals
Can grow in complete absence of oxygen or in air
Lab strains are not pathogenic, but other strains can cause
variety of intestinal diseases
Prototrophic can grow in minimal media
Cells divide every 20 minutes.

Can grow enormous number of cells in short time to examine rare


genetic events.

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Hartwell et al., 5th ed., Chapter 13
Bacteria are monoploid: All mutations
express their phenotype
Altered colony morphology
Large or small; shiny or dull; round or irregular

Resistance to bactericides
Antibiotics, bacteriophages

Auxotrophs unable to reproduce in minimal media


Defective in enzymes required to synthesize complex
compounds (e.g. amino acids, nucleotides)

Defective in using complex chemicals from the environment


Example - breaking down lactose into glucose and galactose

Defective in proteins essential for growth


Conditional lethal mutations, e.g. temperature-sensitive (ts)
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Hartwell et al., 5th ed., Chapter 13
Screens versus selection

Selection establish conditions in which only the desired


mutant will grow
Select for streptomycin resistance (Strr) by plating on media
containing streptomycin

Select for prototrophic revertants by plating auxotrophs on


minimal media

Genetic screen examine each colony for a particular


phenotype

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Hartwell et al., 5th ed., Chapter 13
Gene transfer in bacteria: An overview

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Hartwell et al., 5th ed., Chapter 13
Transformation competent cells can take up
DNA fragments from surrounding environment

Natural transformation happens when bacteria take up DNA


fragments spontaneously from their surroundings.
Artificial transformation can be accomplished in the lab by
making the cells competent
Treat cells with calcium to make the cell walls and
membranes permeable to DNA or use electroporation

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Hartwell et al., 5th ed., Chapter 13
Natural transformation
in B. subtilis

Selection for His+ and/or Trp+ is


used to identify transformants
Then, screen for His+ Trp+ co-
transformants
Genes close together have a higher
frequency of co-transformation than
genes that are further apart

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Hartwell et al., 5th ed., Chapter 13
Selecting and screening for transformation

Selection and screening for gene transfer from His+ Trp+ donor to
His Trp recipient:

To select for Trp+ transformants, plate on minimal media with


histidine and no tryptophan

To select for His+ transformants, plate on minimal media with


tryptophan and no histidine

To screen for His+ Trp+ co-transformants, test Trp+ transformants


and His+ transformants for growth on minimal media with neither
tryptophan nor histidine
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Hartwell et al., 5th ed., Chapter 13
Demonstration of gene transfer by conjugation

Lederberg and Tatum, 1940s

Fig. 13.14
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Hartwell et al., 5th ed., Chapter 13
Bacterial conjugation requires
direct cell to cell contact

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Hartwell et al., 5th ed., Chapter 13
The F plasmid contains genes for synthesizing
connections between donor and recipient cells
Donors for conjugation are F+ (carry an F plasmid)
Recipients for conjugation are F (don't carry an F plasmid)

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Hartwell et al., 5th ed., Chapter 13
Formation of an Hfr chromosome
F plasmid has three IS elements, which are identical to IS
elements found at various positions on the bacterial
chromosome
High frequency recombinant (Hfr) cells are formed when an
F plasmid integrates into the bacterial chromosome through
recombination between IS elements

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Hartwell et al., 5th ed., Chapter 13
Different Hfr
chromosomes
An F plasmid that can
integrate into the bacterial
genome is an episome.

Hfr strains differ in the


location and orientation of the
integrated episomes.

Hfr strains retain all F plasmid


functions and can be a donor
for conjugation with an F
strain.

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Hartwell et al., 5th ed., Chapter 13
Gene transfer between
Hfr donors and F
recipients

Transfer of DNA starts in the F


plasmid at the origin of transfer

Chromosomal genes located


next to F plasmid sequences
are transferred to the recipient

Transferred chromosomal DNA


recombines into homologous
DNA in recipient

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Hartwell et al., 5th ed., Chapter 13 27
Mapping genes by gene transfer
during conjugation
Interrupted-mating
experiment
Genes that
immediately follow
the origin of
transfer in Hfr
chromosome are
transferred first
Order of transfer
reflects the gene
order on the
chromosome

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Hartwell et al., 5th ed., Chapter 13
Formation of F plasmids by excision from an
Hfr chromosome

F plasmid is formed by
excision of F plasmid
plus some adjacent
bacterial chromosomal
DNA
F plasmids replicate
independently in bacterial
cells.

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Hartwell et al., 5th ed., Chapter 13
Formation of F plasmids by excision from an
Hfr chromosome
F plasmids can be transferred to F- cells by conjugation.

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Hartwell et al., 5th ed., Chapter 13
Use of F plasmids for
complementation studies
Conjugation with F plasmids can make partial diploids,
called merodiploids.
If the merodiploid shown can grow without tryptophan, the
two trp- mutations are in different genes.

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Hartwell et al., 5th ed., Chapter 13
Integration of the phage DNA initiates
the lysogenic cycle

Recombination between att sites on phage and the


bacterial chromosome allows integration of the prophage

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Hartwell et al., 5th ed., Chapter 13
Excision of a prophage from a lysogen
Abnormal excision produces a specialized transducing
phage
Bacterial DNA adjacent to integration site can be packaged
with viral DNA and then transferred to a recipient cell

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Hartwell et al., 5th ed., Chapter 13
Identifying a mutant bacterial gene by plasmid
library transformation

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Hartwell et al., 5th ed., Chapter 13
Transposons as mutagens

Mariner transposon
No ORI, only replicates if
inserted into bacterial
chromosome.

Antibiotic resistance gene,


can select for bacteria with a
transposon in chromosome.

Each insertion may disrupt a


different gene

Can screen colonies for


mutant phenotype of interest

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Hartwell et al., 5th ed., Chapter 13
Identifying transposon insertion site
by inverse PCR
Genes interrupted by a
mariner transposon can be
identified easily by inverse
PCR.
PCR primers are
complementary to known
transposon DNA sequence.

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Hartwell et al., 5th ed., Chapter 13
Gene targeting is used to make mutations in
specific genes.
Insert linear DNA
construct into bacteria
drug resistance gene

>50 bp of sequence
homologous to 5 and 3
ends of gene X

Homologous
recombination results in
replacement of gene X
with drug resistance gene.

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Hartwell et al., 5th ed., Chapter 13
13.6 A Comprehensive Example: How N.
gonorrhoeae Became Resistant to Penicillin
Learning objectives:
Explain how penicillin kills bacteria.

Describe mechanisms of penicillin resistance and how N.


gonorrhoeae has become resistant.

Discuss potential solutions to the worldwide problem of drug-


resistant pathogens.

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Hartwell et al., 5th ed., Chapter 13
Penicillin interferes with synthesis of the
bacterial cell wall
Penicillin binds to transpeptidase, inhibits its enzymatic
activity, and prevents cross-linking.

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Hartwell et al., 5th ed., Chapter 13
The penicillin resistance gene (penr)

H. gonorrhoeae acquired a plasmid from H. influenzae that


had the penr gene.
penr encodes penicillinase.

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Hartwell et al., 5th ed., Chapter 13
A second method to become penicillin
resistant: Mutation of chromosomal genes
penA encodes transpeptidase
Mutation decreases affinity for penicillin

penB encodes a porin, a protein in the outer cell wall that


regulates entry into the periplasm
Mutation decreases entry of penicillin to cell

mtr encodes a repressor of an eflux pump


Mutation results in increased pumping of penicillin out of cell.

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Hartwell et al., 5th ed., Chapter 13
Mutations that lead to penicillin resistance

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Hartwell et al., 5th ed., Chapter 13
What should we do about the
problem of drug resistance?
Reduce antibiotic use
Develop novel classes of antibiotics
Make existing antibiotics more effective
Other imaginative ideas?

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Hartwell et al., 5th ed., Chapter 13
PART
How Genes Travel on Chromosomes CHAPTER
IV

Organellar Inheritance

CHAPTEROUTLINE
14.1 Mitochondria and Their Genomes
14.2 Chloroplasts and Their Genomes
14.3 The Relationship Between Organellar and Nuclear Genomes
14.4 Non-Mendelian Inheritance of Mitochondria and Chloroplasts
14.5 Mutant Mitochondria and Human Disease

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Hartwell et al., 5th ed., Chapter 14
Organelle genomes lead to
non-Mendelian inheritance
Four-oclocks have green or variegated leaves.

This trait is inherited from the mother (maternal inheritance), due


to genes found on chloroplast genome.

Mitochondria and chloroplasts are nonnuclear organelles with


their own small genomes.

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Hartwell et al., 5th ed., Chapter 14
14.1 Mitochondria and their genomes

Learning objectives:
Describe the structure and function of a typical
mitochondrion.

List ways in which mitochondrial genomes vary among


different species.

Summarize RNA editing in mitochondria.

Discuss exceptions to the universal DNA code found in


mitochondria.

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Hartwell et al., 5th ed., Chapter 14
Structure and function of mitochondria

Mitochondria Membrane bound cytoplasmic organelles

Many in each eukaryotic


cell

Outer membrane
surrounds wrinkled
inner membrane

Produce energy packets


(ATP) through the
Krebs cycle and
oxidative
phosphorylation

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Hartwell et al., 5th ed., Chapter 14
Human mitochondrial genome

Compact gene arrangement


16.5 kb genome

37 genes endoding tRNAs,


rRNAs, and proteins for
oxidative phosphorylation.

No introns

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Hartwell et al., 5th ed., Chapter 14
Variation in mitochondrial genomes

Vary in size from 6-2400 kb


Some have introns and space
between genes
Some are circular, others linear
Trypanosomes have a
kinetoplast single
mitochondrion with interlocking
circles of mtDNA.

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Hartwell et al., 5th ed., Chapter 14
RNA editing in the kinetoplast

Maxicircles contain genes, but sequence does not exactly


match corresponding cDNAs.
RNA editing
alters pre-mRNA sequence to
make a mature mRNA.

Adds start and stop codons

Changes many new codons

Minicircles encode guide


RNAs
Editosome use guide RNAs
as templates for RNA editing.
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Hartwell et al., 5th ed., Chapter 14
Mitochondrial exceptions to universal code

Mitochondrial genetic code varies in different organisms.


In humans, there are 5 differences between the universal
and mitochondrial genetic codes.

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Hartwell et al., 5th ed., Chapter 14
14.3 The relationship between
organellar and nuclear genomes
Learning objectives:
Describe the cooperation between organellar and nuclear
genomes.

Summarize the endosymbiont theory of organelle origin.

Explain the implications of gene transfer from organellar


genomes to the nucleus.

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Hartwell et al., 5th ed., Chapter 14
Cooperation between nuclear
and organellar genomes
Mitochondria and chloroplasts require nuclear gene
products to assemble and function.
Cytochrome oxidase c
functions in mitochondrial electron transport

7 subunits: 3 encoded by mitochondrial genome, 4 by nuclear


genes

Nuclear genes encode majority of protein required for gene


expression in mitochondria and chloroplasts

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Hartwell et al., 5th ed., Chapter 14
Mitochondria and chloroplasts have
characteristics of prokaryotic cells
Endosymbiont theory mitochondria and chloroplasts are
descended from bacteria that fused with nucleated cells
Have their own DNA

Like in bacteria, mtDNA and cpDNA are not arranged into


nucleosomes.

Mitochondria use N-formyl methionine and tRNAfMet in


translation.

Inhibitors of bacterial translation block mitochondrial and


chloroplast translation, but not eukaryotic translation.

Comparisons of rRNA gene sequences suggest mitochondrial


and chloroplast genomes derive from a common ancestor of
nonsulfur and cyanobacteria, respectively.
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Hartwell et al., 5th ed., Chapter 14
Implications of gene transfer between
organelles and the nucleus
Organelles must function inside the cell
Organelle gene incorporated into nuclear genome

organelle copy of the gene becomes redundant

Changes may make it non-functional

Different organelle genes transferred to nucleus in different


lineages leading to organelle diversity

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Hartwell et al., 5th ed., Chapter 14
14.5 Mutant mitochondria and human disease

Learning objectives:
Recognize mitochondrial diseases in human pedigrees.

Explain the effect of heteroplasmy on the manifestation of


mitochondrial diseases.

Discuss why some scientists believe a relationship exists


between mitochondria and aging.

Describe how oocyte nuclear transfer might be used in the


future to prevent transmission of mitochondrial disease.

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Hartwell et al., 5th ed., Chapter 14
Characteristic pedigree for mitochondrial
disease.
Several diseases of the human nervous system are caused
by mutations in the mitochondrial genome.
Mutations are passed from mothers to children.
Symptoms vary due to heteroplasmy

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Hartwell et al., 5th ed., Chapter 14
mtDNA mutations and human health

Maternal pattern of inheritance

Myoclonic epilepsy and ragged red fiber disease (MERRF)


Range of symptoms
Mutations in mitochondrial tRNAs (e.g. tRNALys)
Deleterious effect on ATP production
Individuals affected by MERFF are heteroplasmic
Severity of phenotype depends on percentage of mutant
mtDNA

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Hartwell et al., 5th ed., Chapter 14
Tissue distribution of mutant mitochondria
and MERFF phenotype

Tissues with higher


energy requirements are
less tolerant of mutant
mitochondria

Tissues with low energy


requirements are affected
only when the proportion
of wild-type mitochondria
is greatly reduced

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Hartwell et al., 5th ed., Chapter 14
Mitochondrial mutations may have
an impact on aging
Oxidative phosphorylation system in the mitochondria
generates free radicals, which can damage DNA
Accumulation of mtDNA mutations over time may result in
age-related decline in oxidative phosphorylation
Evidence in support of role of mtDNA and aging:
Percentage of heart tissue with a mitochondrial deletion
increases with age

Brain cells of people with Alzheimers disease (AD) have


abnormally low energy metabolism

20% to 35% of mitochondria in brain cells of most AD


patients have mutations in cytochrome c oxidase genes,
which may explain the low energy metabolism
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Hartwell et al., 5th ed., Chapter 14
Oocyte nuclear transplantation can sidestep
transmission of mitochondrial disease

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Hartwell et al., 5th ed., Chapter 14
Oocyte nuclear transfer is
successful in primates

Mito and Tracker


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Hartwell et al., 5th ed., Chapter 14

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