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Chapter 9: Gene Transfer, Mutations, and Genome

Evolution

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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-positive bacteria transform DNA using a transformasome complex.

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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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Relaxosome: many genes necessary for DNA transfer (halicase, endonuclease, etc.

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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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Specialized Transduction

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Specialized 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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Figure 9.9

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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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Homologus DNA

Recombinants

Crossingover

Recombination

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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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Missense mutation: Changes the amino acid sequence to another

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Nonsense mutation: Changes the amino acid sequence to a stop codon

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Frame-shift mutation: Changes the open-reading frame of the gene

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Mutation due to inversion in DNA strands

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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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3) Formation of apurinic sites [loss of purine]

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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

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Mutagenic agents and their effects.

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UV radiation can induce dimerization