Transcript Gene transfer in bacteria - McGraw Hill Higher Education

PowerPoint to accompany
Genetics: From Genes to
Genomes
Fourth Edition
Leland H. Hartwell, Leroy Hood,
Michael L. Goldberg, Ann E. Reynolds,
and Lee M. Silver
Prepared by Mary A. Bedell
University of Georgia
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PART
IV
How Genes Travel on Chromosomes
CHAPTER
Prokaryotic and
Organelle Genetics
CHAPTER OUTLINE



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14.1 A General Overview of Bacteria
14.2 Bacterial Genomes
14.3 Gene Transfer in Bacteria
14.4 Bacterial Genetic Analysis
14.5 The Genetics of Chloroplasts and Mitochondria
14.6 Non-Mendelian Inheritance of Chloroplasts and Mitochondria
14.7 mtDNA Mutations and Human Health
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Studies of bacteria were critical to the
development of the field of genetics
Classical bacterial genetics – 1940s to 1970s
• Virtually all knowledge of gene structure, expression,
and regulation came from studies of bacteria and
bacteriophages
Advent of recombinant DNA technology – 1970s and 1980s
• Depended on understanding of bacterial genes,
chromosomes and restriction enzymes
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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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Bacteria sizes and characteristics
Vast range of sizes
• Smallest has 200 nm diameter
• Largest is 500 μm in length
All bacteria are prokaryotes, which lack a defined nuclear
membrane
All bacteria lack membrane-bounded organelles
Bacterial chromosomes fold to form a nucleoid body that
excludes ribosomes
Most bacteria have a cell wall made of carbohydrate and
peptide polymers that surrounds the cell membrane
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Metabolic diversity of bacteria
Play essential roles in many natural processes
• Balance of microorganisms is key to success of ecological
processes that maintain the environment
Nitrogen cycling
• Decomposing bacteria break down plant and animal matter
and produce ammonia (NH3)
• Nitrifying bacteria use NH3 as source of energy and release
nitrate (NO3), which is used by some plants
• Denitrifying bacteria convert nitrate into atmospheric
nitrogen (N2)
• Nitrogen-fixing bacteria live in roots of some plants and
convert N2 to ammonium (NH4+) for their host plant to use
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Bacteria must be grown and studied in cultures
Bacteria grown as
a cell suspension
in liquid media
Bacteria grown as colonies
on solid nutrient-agar in a
petri dish
Fig. 14.2
Fig. 14.1
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Escherichia coli (E. coli) is
a versatile model organism
E. coli is the most studied and best understood bacterial
species
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
• Single carbon and energy source (e.g. glucose)
• Inorganic salts
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Examples of phenotypic variation
in bacteria due to mutations
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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Finding mutations in bacterial genes
Rapid bacterial multiplication allows detection of very rare
genetic events
• In minimal media, bacteria divide every 60 min
• In rich media, bacteria divide every 20 minutes
Effectively haploid – straightforward relationship between
mutation and phenotypic variation
Selection – establish conditions in which only the desired
mutant will grow
• e.g. Select for streptomycin resistance (Strr) by plating on
media containing streptomycin, select for prototrophic
revertants by plating auxotrophs on minimal media
Screen – examine each colony for a particular phenotype
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Techniques used to identify rare mutants
Spontaneous mutations in specific bacterial genes occur
very rarely (1 in 106 to 1 in 108)
Replica plating – simultaneous transfer of thousands of
colonies from one plate to another (see Fig 7.5a)
Mutagens – used to increase the frequency of mutations
(see Fig 7.10)
Enrichment – increases the proportion of mutant cells
• e.g. Penicillin kills only cells that are dividing but not cells
that are unable to divide (Fig. 14.4)
Testing for visible phenotypes
• e.g. β-galactosidase from wild-type lacZ gene breaks down
X-Gal substrate into a blue pigment
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Penicillin enrichment for auxotrophic mutants
Fig. 14.4
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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
Fig. 14.5
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The E. coli genome was sequenced in 1997
Genome of K12 strain was
sequenced
4.6 Mb
~ 90% of genome encodes
protein
4288 genes, but function known
for only 60%
On average, 1 gene per kb
No introns
Very little repetitive DNA
Small intergenic regions
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Fig. 14.6
14
Insertion sequence (IS) elements dot the
genomes of many types of bacteria
Bacterial strains have different numbers and distributions of
IS elements
• e.g. 15-25 in E. coli, none in B. subtilis
Small transposable elements (700-5000 bp length)
• Inverted repeats (IRs) at ends
• Carry transposase gene
• Can move to other locations in genome
• Can disrupt genes by insertion into coding regions (Fig
14.7b)
Fig. 14.7a
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Tn elements in bacteria are composite
transposable elements
Contain transposase gene and genes conferring resistance
to antibiotics or toxic metals (e.g. mercury)
• e.g. Tn10 – two different IS elements flank 7 kb of DNA that
includes a gene for tetracycline resistance
Easily scored marker for genetic analysis (gene disruptions
and mapping experiments) and for transferring a disrupted
gene to another strain
Fig. 14.8
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Identifying a gene that was disrupted by
insertion of a Tn element
Fig. 14.9
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Genomic analyses in bacteria have created
an information explosion
Complete genome sequence known for hundreds of
prokaryotic species and partial genome sequence known
for thousands of species
New avenues of research are possible with genome studies
• Metagenomics – analysis of genomic DNA from a community
or habitat
 Microbial ecology and communities - DNA sequencing of bacteria
in extreme and unusual environment (Fig. 14.10)
• Comparative genome analysis – identify similarities and
differences between genomes of different species
• Genome studies and public health – aid in development of
vaccines, identify new drug targets, identify specific bacterial
strains in epidemiological studies
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New analyses for assessing microbial diversity
Fig.
14.10
Bacteria in extreme environments are difficult to culture in lab
Rapid DNA sequencing, large-scale PCR amplification, and DNA
arrays can be used to survey composition of microbial
communities and metabolic status
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Plasmids are smaller circles of DNA that carry
genes beneficial to the host cell
Plasmids vary in size from 1 kb to several Mb in length
Plasmids don't carry genes that are essential to the host
Examples of plasmid genes that are beneficial to the host
• Genes that protect host against toxic chemicals (e.g.
mercury) and metabolize environmental pollutants (e.g.
toluene, napthalene, petroleum products)
• Pathogenic genes (e.g. toxins produced by S. dysenteriae)
• Genes encoding resistance to antibiotics
• Multiple antibiotic resistance often due to composite IS/Tn
elements on a plasmid (see Fig. 14.12)
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Some plasmids contain multiple antibiotic
resistance genes and transposons
Movement of antibiotic resistance genes to the plasmid was
facilitated by transposons
Multiple antibiotic resistance genes can be transposed from
the plasmid as a unit
Fig. 14.12
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Gene transfer in bacteria
Lateral (or horizontal) gene transfer – traits are introduced
from unrelated individuals or from different species
• Vertical gene transfer occurs in sexually reproducing
organisms – traits are transferred from parent to
offspring
Three mechanisms for gene transfer in bacteria (Fig. 14.13)
• In all three mechanisms:
 Donor bacterium provides the DNA that is transferred
 Recipient bacterium receives the DNA, which can result in
altered phenotype
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Gene transfer in bacteria: An overview
Fig. 14.13
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In transformation, the recipient takes up DNA
that alters its genotype
Transformation – competent cells can take up DNA
fragments from surrounding environment
Natural transformation occurs in some bacterial species
• e.g. B. subtilis, S. pneumoniae (Griffith's experiments, see
Chapter 6), H. influenzae, N. gonorrhoeae
• In B. subtilis, competence occurs only in nearly starved cells
at specific times in growth culture
 1% - 5% of cells become competent
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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Natural transformation
in B. subtilis
Selection for His+ and/or Trp+ is
used to identify transformants
Then, screen for His+ Trp+ cotransformants
Genes close together have a higher
frequency of co-transformation than
genes that are further apart
Fig. 14.14
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Selecting and screening for transformation
Selection and screening for gene transfer from His+ Trp+ donor to
His− Trp− recipient:
Fig. 14.14a
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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Demonstration of gene transfer by Joshua
Lederberg and Edward Tatum (late 1940s)
This type of gene transfer requires direct cell-to-cell contact
and was later shown to be conjugation
Fig. 14.15
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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)
Fig. 14.16a
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The process of conjugation
Fig. 14.16b
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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
Fig. 14.17
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Different Hfr
chromosomes
20-30 different Hfr strains can
be generated that differ in the
location and orientation of the
integrated F plasmids
Hfr strains retain all F plasmid
functions and can be a donor
for conjugation with an F−
strain
Fig. 14.18
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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
Fig. 14.19
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Interrupted-mating experiments
with Hfr and F− strains
Genes closest to origin
of transfer in F plasmid
are transferred first
Order of transfer
reflects the gene order
on the chromosome
Fig. 14.20
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Mapping genes by interrupted-mating
with Hfr and F− strains
(a) Time of gene transfer
(b) Map based on mating
results
Fig. 14.21
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In transduction, a phage transfers DNA from a
donor bacterium to a recipient bacterium
Bacteriophages (aka phages) are viruses that infect,
multiply in, and kill various species of bacteria
• Widely distributed in nature
• Most bacteria are susceptible to one or more phages
Transduction – process by which a phage transfers DNA
from one host cell to another host cell
Virulent phages – always enter lytic cycle after infecting
cell, multiply rapidly, and kill cell
Temperate phages – can enter either lytic cycle or enter an
alternative lysogenic cycle
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The lytic cycle of phage multiplication
Lytic cycle - results in cell lysis and release of progeny
phage
• Phage injects its DNA into bacterial cell
• Phage proteins are expressed and take over protein
synthesis and DNA replication machinery of infected cell
• Phage DNA replication occurs
• Phage particles are assembled with phage DNA and phage
protein
• Infected cell bursts (lyses) and releases 100-200 new viral
particles able to infect other cells
Lysate – the population of phage particles released from
host bacteria at the end of the lytic cycle
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Generalized transduction
Incorporation of random fragments
of bacterial DNA from donor into
bacteriophage particles
DNA from donor cell injected into
infected recipient cell
Transduced chromosomal DNA
recombines into homologous DNA
in recipient
Fig. 14.22
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Mapping genes
by cotransduction frequencies
~90 kb of DNA (~ 2% of genome) can be transduced
Frequency of cotransduction is higher for genes that are
close together compared to genes that are further apart
Fig. 14.23
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Temperate phages
Choice of lytic or lysogenic cycle (Fig 14.24) depends on
many factors, including environmental conditions
Lysogen – bacteria that harbor an integrated temperate
phage
Prophage – temperate phage that has integrated into host
chromosome
Bacteriophage lambda (λ) is the most commonly used
temperate phage
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Lytic and lysogenic modes of reproduction
Prophages:
Do not produce the viral proteins
needed for more virus particles
Lysogens can be induced to enter
lytic cycle
Fig. 14.24
• Prophage excises from
chromosome, undergo replication,
form new virus particles
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Integration of the phage DNA initiates
the lysogenic cycle
Recombination between att sites on phage λ and the
bacterial chromosome allows integration of the prophage
Fig. 14.26a
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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
Fig. 14.26b
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Comparison of generalized and
specialized transduction
When donor DNA is packaged by phage:
• Generalized transduction – during lytic cycle
• Specialized transduction – during transition from
lysogenic to lytic cycles
Which donor DNA can be packaged with phage:
• Generalized transduction – any bacterial gene or set of
genes on the correct size of DNA fragment
• Specialized transduction – only those bacterial genes
near the integration site of the phage
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Evolutionary implications of
lateral gene transfer
Genomic analysis has revealed widespread occurrence of
gene transfer mechanisms in many bacterial species
Gene transfer is an important mechanism for rapid
adaptation to environmental changes and to development of
pathogenic strains of bacteria
• e.g. presence of diptheria toxin of Corynebacterium
diphtheriae on a lysogenic phage
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Genomic islands originated from
transfer of foreign DNA
Large segments of DNA (10-200 kb in size)
• G+C content is different from the rest of the genome
• Presence of direct repeats at each end
• Found at sites where tRNAs genes are located
• Contain integrase genes and sites for integration
Pathogenicity islands are a subtype of genomic islands
• Lateral transfer of a "package" of genes from a pathogenic
species to a nonpathogenic species
Fig. 14.27
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Pathogenicity islands in Vibrio cholera
Variation in genes present in pathogenicity islands of
different strains of V. cholera
Severity of cholera epidemic depends on genes present in
the strain
• Enterotoxin interferes with host-cell function
• Invasion proteins for travel of bacterium through mucus of
the intestinal tract
• Pilus formation to allow phage attachment
• Phage-related integrases
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Pathogenicity islands in E. coli
Integrative and conjugative elements (ICE) allow transfer of
DNA between many different species
• Have features of conjugative plasmids (like F factor),
encodes an integrase (like lambda phage) and machinery for
conjugation
One pathogenic E. coli strain has an ICE that is similar to
Yersinia pestis and Y. pseuodotuberculosis
• Contains genes for mating pair formation, presumed origin
of transfer, integrase for excision of the element
E. coli strain O157:H7 – causes diarrhea or meningitis
• Encodes proteins for attachment to epithelial cells, changes
to cytoskeleton, loss of fluid, toxin from Shigella that
damages kidneys and intestines
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Bacterial genetic analysis
Transposons allow manipulation of bacterial genomes
• Useful mutagenic agents because they can disrupt genes
and carry genes for antibiotic resistance
Reverse genetics provides a way to insert synthetic genes
to test function
• Recombineering (Fig. 14.28) - replacement of a wild-type
gene with a knockout gene through in vivo recombination
Genomic and genetic approaches can be combined
• Create large scale mutant library by random transposon
mutagenesis, identify sites of insertion by PCR amplification
and comparing sequence to genome sequence
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Recombineering
(a) In vitro: Use recombinant
DNA techniques to create a
defective allele of a gene by
insert of a selectable marker
(e.g. antibiotic resistance)
(b) In vivo: Introduce DNA
fragment into cells, induce
recombination, and select
for antibiotic resistance
Fig. 14.28
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Mitochondria and chloroplasts in eukaryotes
have characteristics of prokaryotic cells
Endosymbiotic theory – mitochondria and chloroplasts are
descended from bacteria that fused with nucleated cells
Mitochondria – organelles that produce energy for
metabolic processes, found in all eukaryotic cells
•Each cell has many mitochondria, highest number in cells with
high energy requirements
•Similar in size and shape to modern aerobic bacteria
•Produces energy in the form of ATP
Chloroplasts – organelles that capture energy from light and
store it as carbohydrates, found in plant and algal cells
•Structural similarities to certain cyanobacteria
•40-50 per cells
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The genomes of mitochondria (mtDNA)
Located within highly condensed structures (nucleoids)
• Number of nucleoids per mitochondria varies depending on
growth conditions and energy needs of cell
mtDNA replication occurs independent of cell cycle
• Random occurrence of replication – some mtDNAs replicate
many times, and other mtDNAs don't replicate at all
In most species, mtDNA is circular
• Some species (Tetrahymena, Paramecium, Chlamydomonas,
Hansenula) have linear mtDNA
• Protozoan parasites have single mitochondrion (kinetoplast)
with large network of minicircles and maxicircles (Fig. 14.30)
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The size and gene content of mitochondrial
genomes varies from organism to organism
Table 14.1
Table 14.2
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Mitochondrial genome variation across species
Mitochondrial genome in humans is very compact
• Adjacent genes either abut each other or overlap slightly
• Virtually no intergenic regions
• No introns
Mitochondrial genome of S. cerevisiae is 4X larger than in
humans and other animals
• Long intergenic regions
• Has introns
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Editing of RNA transcripts
RNA editing first discovered in mtDNA transcripts of
trypanosomes (a protozoan parasite)
• Transcription of mtDNA produces pre-mRNA that is
converted to mature mRNAs by RNA-editing
• RNA editing produces start and stop codons for translation
as well as internal codons
RNA editing also identified in some plants and fungi
Fig. 14.31
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Mitochondrial translation differs from
translation of mRNAs from nuclear genes
Similar aspects of translation in prokaryotes
• Initiation of translation by N-formyl methionine and tRNAfMet
• Translation in prokaryotes and mitochondria is inhibited by
chemicals (e.g. chloramphenicol and erythromycin) that
don't affect translation of nuclear mRNAs
The genetic code for
nuclear and
mitochondrial genes
is different
Table 14.3
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The genomes of chloroplasts (cpDNA)
Includes genes for some photosynthetic enzymes and for
gene expression
Ranges in size from 120-217 kb, but most are 120-160 kb
(see Table 14.4)
Closely packed genes with little intergenic sequence (like
human mtDNA) but has introns (like yeast mtDNA)
Similarities to bacteria
• RNA polymerases of choloroplasts and bacteria are similar
• Translation in prokaryotes and chloroplasts is inhibited by
chemicals (e.g. chloramphenicol and streptomycin) that
don't affect translation of nuclear mRNAs
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Potential uses of transformed chloroplasts
Techniques for introducing genes into organelles
• Gene gun – coat 1 μm metal particles with DNA and then
"shoot" the DNA into cells
• Biolistic transformation – DNA released from particle, enters
nucleus or organelle, recombines into the genome
Can alter properties of commercially important crop plants
• Herbicide resistance
 Maternal inheritance (not through male pollen) limits risk of
escape
• Turn chloroplasts into protein-production factories (i.e. for
vaccines)
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Nuclear and organellar genomes
cooperate with each other
Assembly and maintenance
of functional organelles
depend on both organelle
and nuclear gene products
• e.g. the 7 subunits of
cytochrome c oxidase in
most organisms are
encoded by 3 mitochondrial
genes and 4 nuclear genes
Organelles don't carry all the
genes needed for translation
(semiautonomous)
Fig. 14.32
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Number and genomic location of
oxidative phosphorylation genes
Fig. 14.32
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Gene transfer between organelles
and the nucleus
Evidence for transfer via an RNA intermediate
• COXIII gene in plants – encodes component of mitochondrial
electron transport chain
• Present in the nuclear genomes of some plants but in the
mitochondrial genomes of other plants
• Some plant species have a non-functional mitochondrial
gene that contains an intron and the functional nuclear gene
doesn't have the intron
Evidence for transfer at the DNA level
• Some plant mtDNAs contain large fragments of cpDNA
• Nonfunctional, partial copies of organellar genes are present
in the nuclear genome
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High rate of mutation in mtDNA
mtDNA evolves ~10X more rapidly than does nuclear DNA
• More errors in replication and less efficient repair
Provides valuable tool for studying evolutionary
relationships of closely-related species
But, has little value for studying evolutionary relationships
of distantly-related species
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Non-Mendelian inheritance of organelles
1909 – green vs. variegated leaves in four-o'-clocks
• Variegated offspring produced when ovules of variegated
plants were fertilized with pollen from green plants
• No variegated offspring produced when ovules of green
plants were fertilized with pollen from variegated plants
1949 – when S. cerevisiae grown on glucose, 95% of
colonies were large (grande) and 5% were small (petite)
• Mating grande x grande produced grande diploid,
sporulation produced 4 grande spores
• Mating petite x petite produced petite diploids, but they were
defective for respiration and couldn't sporulate
• Mating grande x petite produced grande diploids, sporulation
produced 4 grande spores and 0 petite spores
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Maternal inheritance of
Xenopus mtDNA
Two closely-related species
of frogs
DNA probes from mtDNA
were used to identify mtDNA
present in F1 offspring
Fig. 14.34
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A maternally inherited neurodegenerative
disorder in humans
Leber's hereditary optic neuropathy (LHON)
G-to-A substitution in gene for an NADH subunit causes
Arg-to-His missense substitution
Fig. 14.35
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Hartwell et al., 4th edition, Chapter 14
64
Distribution of organelles during mitosis
Heteroplasmic cells contain a mixture of organelle genomes
Homoplasmic cells contain only one type of organelle
genome
Mitotic progeny of homoplasmic cells are also homoplasmic
Mitotic progeny of heteroplasmic cells can be either
heteroplasmic, homoplasmic wild-type, or homoplasmic
mutant
Uneven distribution of organellar genomes has distinct
phenotypic consequences
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Some organisms exhibit biparental inheritance
of organellar genomes
1909 – reciprocal crosses between green and variegated
geraniums
• Both types of crosses produces green, white, and variegated
offspring in varying proportions
• Chloroplast traits inherited from both parents
Fig. 14.37
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mtDNA mutations and human health
Maternal pattern of inheritance
Symptoms vary enormously among family members
Myoclonic epilepsy and ragged red fiber disease (MERFFF)
• Range of symptoms: uncontrolled jerking, muscle weakness,
deafness, heart problems, kidney problems, progressive
dementia
• Mutations in mitochondrial tRNAs (e.g. tRNALys)
• Disruption of mitochondrial transport chain
• Individuals affected by MERFF are heteroplasmic
• Severity of phenotype depends on percentage of mutant
mtDNA (see Fig. 14.39 and 14.40)
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The proportion of mutant mitochondria
determines the severity of the MERFF phenotype
and the tissues affected
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
Fig. 14.40
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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 Alzheimer’s 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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