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Chapter Overview
● The mosaic nature of genomes
● Gene transfer: Transformation; conjugation; and transduction
● Genetic recombination
● Mutations: Types and causes
● Mechanisms of DNA repair
● Mobile genetic elements
- Insertion sequences and transposons
● How genomes evolve
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Introduction
DNA sequences change over generations through various mutations, rearrangements, and inter- and intraspecies gene transfer.
But what are the consequences of DNA plasticity?
This chapter explores long-standing evolutionary questions and shows how microbial genomes continually change.
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A surprise arising from bioinformatic studies is the mosaic nature of all microbial genomes.
- For example, E. coli’s genome is rife with genomic islands, inversions, deletions, and paralogs and orthologs
- This is the result of heavy horizontal gene transfer, recombinations, and a variety of mutagenic and DNA repair strategies.
The Mosaic Nature of Genomes
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In bacteria recombination occurs in a number of ways:
• Transformation: Free DNA is transferred• Transduction: DNA transfer via a virus• Conjugation: Cell-to-cell contact and a
plasmid is involved.
Recombination: Mechanisms of Genetic Transfer
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Gene Transfer by Transformation
Transformation is the process of importing free DNA into bacterial cells.
- the cells need to be competent.
Many cells are capable of natural transformation and naturally competent.
-others require artificial manipulations.
- Perturbing the membrane by chemical (CaCl2) or electrical (electroporation) methods
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• Not all bacteria can take up free or naked DNA (<1%).
• Some microbes become competent sometime during their growth cycle
Gene Transfer by Transformation
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Natural Transformation occus Bacillus sp., Haemophilus sp., Neisseria sp., Acinetobacter sp., Streptococcus sp., Pseudomonas sp.
Gene Transfer by Transformation
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Gram-negative bacteria transform DNA without the use of competence factors (CF).
• some Gram negative organisms are always competent or they become competent when starved.
• also, they do not use transformasomes. • most Gram-negative species is sequence-
specific.
Thus limiting gene exchange between genera
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Conjugation (mating)
Conjugation involves a cell-to-cell contact mediated by a special plasmid, conjugative plasmid
• Gram Negative: The plasmid carries genes that code for a sex-pilus
• Gram Positive: Sticky molecules help bind two cells together.
• Gram Negative Bacteria with conjugative plasmids are males and without it are females
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Gene Transfer by ConjugationConjugation is the transfer of DNA from
one bacterium to another, following cell-to-cell contact by pilus on the donor cell.
- The pilus attaches to the receptor on the recipient cell
- Two cell fuse and single-stranded DNA passes from donor to recipient cell.
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Conjugation requires the
presence of special
transferable plasmids
(conjugative plasmids).
A well-studied example in E. coli is the fertility factor (F factor). Also called fertility plasmid
Conjugation begins with contact between the donor cell, called the F+ cell, and a recipient F– cell.
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• The cells with an unintegrated conjugative plasmidare called F+ (males) and cells that act as recipient for F+ are F- (Females)
• When a donor with F+ plasmid transfers a copy of the plasmid to a recipient (F-), the recipient becomes F+
Conjugation
F+ + F- F+ + F+
Conjugation
Female cells become male cells and be able to transfer the plasmid
Conjugation
Figure 8.27a
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Conjugation
The F-factor plasmid can integrate into the chromosome.
- The cell is now designated Hfr, or high-frequency recombination strain.
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Conjugation betweenan Hfr and F-, the recipient gets some of the Hfr genesplus some of the donor’s genes. The recipient becomes a recombinant F-, since not all Hfr genes are transferes.
The entire chromosome take about 100 min to transfer as opposed only 5 min for free plasmid
ConjugationHfr + F- Hfr + F-
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An integrated F-factor can excise from the chromosome.- Aberrant excision results in an F′ factor or F′ plasmid, which carries chromosomal genes.
Figure 9.5
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Some bacteria can actually transfer genes across biological domains.
Transfer of Genes into Eukaryotes
- Agrobacterium tumefaciens, which causes crown gall disease
- Contains a tumor-inducing plasmid (Ti) that can be transferred via conjugation to plant cells
Figure 9.6
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Gene Transfer by Transduction
Transduction is the process in which bacteriophages carry host DNA from one cell to another.
There are two basic types:
- Generalized transduction: Can transfer any gene from a donor to a recipient cell
- Specialized transduction: Can transfer only a few closely linked genes between cells
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Generalized Transduction
Any gene from a donor chromosome is packaged into a bacteriophage and transferred to a new cell upon infection.
Salmonella enterica
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• Bacteriophases with a foreign DNA are called transducing particles.
•The transducing particles transfer any part of the host DNA to a new host (recipient) cells.
•Recombination occurs at low frequency
P1 phage of E.Coli. and P22 phage of Samonella are examples of generalized transduction.
Steps of generalized transduction
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• The Phage genome is integrated into the host DNA at a specific site.
• On induction (UV light), the viral DNA separates from the host genome.
• Under rare events, the phage DNA maybe excised incorrectly.
• Some of the adjacent bacterial genes are excised along with the viral genome.
• When the phage infects new crop of cells, it allows transduction to occur at high frequency
Specialized Transduction
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Bacteria have developed a kind of “safe sex” approach to gene exchange.
This protection system, called restriction and modification, involves:
- Enzymatic cleavage (restriction) of alien DNA, by restriction endonucleases
- Protective methylation (modification) of host DNA
DNA Restriction and Modification
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RecombinationTwo different DNA molecules in a cell can
recombine by one of several mechanisms:
- Generalized recombination requires that the two recombining molecules have a considerable stretch of homologous DNA sequences (>50 bp).
- Site-specific recombination requires very little sequence homology between the recombining DNA molecules.
- But it does require a short sequence recognized by the recombination enzyme
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RecA proteins or Synaptases play critical role in recombination
-double stranded DNA becomes single-stranded DNA by creating a nick
-DNA unwinds
-single-stranded binding proteins bind to the ssDNA
-RecA finds homology and mediated strand invasion
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A mutation is a heritable change in the DNA.
Mutations can come in several different forms:
Types of Mutations
- Point mutation: Change in a single base
- Insertion (addition) and deletion (subtraction) of one or more bases
- Inversion: DNA is flipped in orientation
- Reversion: DNA mutates back to original sequence
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Mutations can be categorized into several information classes:- Silent mutation: Does not change the amino acid sequence
DNA template TTT point mutation T TCDNA coding AAA AAGm-RNA UUU UUCAmino acid Phenylalanine Phenylalanine
Though DNA strand has changed, the protein sequence is the same
Mutations
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Spontaneous mutations are rare because of the efficiency of DNA proofreading and repair pathways.
However, they can arise for many reasons:
1)Tautomeric shifts in DNA bases that alter base-pairing properties [ GT or A C]
2) Oxidative deamination of bases
Mutations Arise in Diverse Ways
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Mutations can be caused by mutagens:
Chemical agents- Base analogs- Base modifiers- Intercalators
Electromagnetic radiation- X-rays and gamma rays: Break the DNA- Ultraviolet rays: Form pyrimidine dimers
Mutations Arise in Diverse Ways