Lecture 2: Horizontal Gene Transfer & Mobile Genetic Elements 🧬↪

Learning Objectives:

  • Identify different types of transposons (with examples).

  • Explain the two main models of transposition.

  • Detail the genetic rearrangements produced by transposons.

  • Understand what integrons are and how they work to build using gene cassettes.

Overview of Transposable Elements (Transposons)

  • Transposons are DNA elements that can 'hop' or transpose from one piece of DNA (donor DNA) to another piece of DNA (target DNA).

  • The movement is called transposition.

  • The enzyme that promotes transposition is called a transposase.

  • Transposition is a tightly regulated process.

Common Types of Bacterial Transposons:

  • Insertion Sequence (IS) Elements

  • Composite Transposons

  • Non-Composite Transposons

Models for Transposition

Most bacterial transposons transpose by one of two main mechanisms:

  • Replicative Transposition ("Copy & Paste"):

    • The entire transposon replicates during transposition.

    • This results in two copies of the transposon (one in the original location, one in the new location).

  • Non-Replicative Transposition ("Cut & Paste" or Conservative Transposition):

    • The transposon is removed from one place and inserted into another.

    • The donor DNA may be degraded if the transposon is removed without repair.

Mechanism of Replicative Transposition:

  1. Site-Specific Cleavage: Single-stranded cuts are made on opposite strands at each end of the transposon in the donor plasmid. A pair of single-strand cuts are made at the target sequence in the recipient plasmid.

  2. Ligation & Replication Fork Formation: Each free end of the transposon is ligated to a protruding single strand at the insertion site in the target DNA. This forms a replication fork at each end of the transposon.

  3. Replication: The transposon is replicated, creating a cointegrate structure, which is a fusion of the donor and recipient plasmids, now both containing a copy of the transposon.

  4. Resolution: A site-specific recombination event occurs between internal resolution sites (res sites) within the two copies of the transposon in the cointegrate. This resolves the cointegrate into two separate plasmids, each containing one copy of the transposon. The donor plasmid is restored, and the recipient plasmid now contains the transposon and a duplicated target sequence.

Mechanism of Non-Replicative Transposition:

  • This mechanism is used by many IS elements and composite transposons.

  • The transposase makes double-stranded breaks at the ends of the transposon in the donor DNA.

  • The transposon is excised and inserted into the target DNA at the site of a staggered break (cuts made by the transposase in the target DNA at slightly different positions on each strand).

  • The gaps created by the staggered cut in the target DNA are filled in by DNA repair mechanisms, resulting in a short direct repeat of the target sequence flanking the newly inserted transposon.

  • For most types, removal of the transposon from the donor DNA will result in the degradation of the donor DNA if the break is not repaired. Both strands of the transposon move to the recipient DNA.

Genetic Rearrangements Caused by Transposons

Transposons are nature's genetic tools and can promote various rearrangements of host DNA:

  • Inversions:

    • Can occur when the host DNA contains two copies of a transposon in an inverted orientation relative to each other.

    • Recombination between these inverted transposons inverts the DNA segment located between them.

  • Deletions:

    • If two transposons in the host DNA have the same orientation, recombination between them deletes the segment of DNA located between the transposons.

  • Rearrangement (Relocation):

    • The deletion of a chromosomal segment (as described above), followed by its integration into the chromosome at a different site by a separate recombination event.

Effects of Transposons on Other Genes

Transposon insertion can have various effects on nearby genes:

  1. Insertion outside the gene: If a transposable element inserts outside the coding or regulatory regions of a gene (Gene X), the expression of Gene X may not be affected.

  2. Insertional inactivation: If a transposable element inserts within a gene (Gene X), it can inactivate the gene.

  3. Insertional activation: If a transposable element containing a promoter inserts near a gene (Gene X), it can activate that gene or alter its expression pattern, leading to expression under different conditions than usual.

Types of Bacterial Transposons

1. Insertion Sequence (IS) Elements:

  • Smallest and simplest transposons, typically 750-3,200 bp in length.

  • Over 700 IS elements have been discovered in bacteria.

  • They are normal constituents of bacterial chromosomes and plasmids. For example, E. coli typically contains multiple copies of IS1, IS2, IS3, and IS4.

  • An IS element usually only codes for the transposase enzyme and is flanked by short inverted repeat (IR) sequences.

  • Families of IS Elements (based on transposase type):

    • DDE Transposases: Contain conserved Aspartate (D), Aspartate (D), and Glutamate (E) residues in their active site. They use an OH group as a nucleophile. This is the most abundant type.

    • HUH (or Y) Transposases: Contain a conserved pair of Histidine (H) residues separated by a large hydrophobic amino acid (U). They use Tyrosine (Y) as a nucleophile.

2. Composite Transposons:

  • Consist of a gene-coding central region (often carrying genes for antibiotic resistance or other functions) flanked by two IS or IS-like elements.

  • The IS elements are identical or nearly identical.

  • The flanking IS elements can be in the same orientation (e.g., Tn9, which has direct repeats of IS1) or in an inverted orientation (e.g., Tn5 flanked by inverted IS50 elements; Tn10 flanked by inverted IS10 elements).

  • Examples:

    • Tn5: ~5,700 bp, carries Kanr (kanamycin resistance), flanked by 1,500 bp IS50 elements.

    • Tn9: ~2,638 bp, carries Camr (chloramphenicol resistance), flanked by 768 bp IS1 elements in direct orientation.

    • Tn10: ~9,300 bp, carries Tetr (tetracycline resistance), flanked by 1,400 bp IS10 elements.

3. Non-Composite Transposons (or TnA-family transposons):

  • Consist of a gene-coding central region (carrying genes like transposase, resolvase, and often antibiotic resistance) flanked by two short inverted repeats (IRs). They do not have complete IS elements at their ends.

  • They often transpose replicatively and include a resolution site (res) for resolving the cointegrate intermediate.

  • Examples:

    • Tn3: Carries Ampr (ampicillin resistance), transposase (tnpA), and resolvase (tnpR) genes.

    • Tn501: Carries Merr (mercury resistance) genes.

    • γδ (gamma-delta transposon): Structurally similar to Tn3.

Transposable Elements in Eukaryotes

Eukaryotic transposable elements are broadly classified:

  • DNA transposons (Class 1 & 2 in Wicker et al. classification):

    • Have short inverted repeats at each end.

    • Contain a gene for transposase.

    • Mode of transposition: Either conservative ('cut & paste', Class 1) or replicative ('copy & paste', Class 2).

    • Examples: P-element & Mariner (Drosophila), Ac-Ds (maize), Tc1 (C. elegans).

  • LTR retroviral-like retrotransposons:

    • Have directly repeated long terminal repeats (LTRs) at their ends.

    • Contain a gene for reverse transcriptase.

    • Mode of transposition: Replicative via an RNA intermediate produced by a promoter in the LTR.

    • Examples: Copia (Drosophila), Ty1 (Saccharomyces cerevisiae), Bs1 (maize).

  • Non-retroviral retrotransposons (also known as non-LTR retrotransposons):

    • Have a poly-A sequence at the 3’-end of the RNA transcript; the 5’-end is often truncated.

    • Contain a gene for reverse transcriptase.

    • Mode of transposition: Replicative via an RNA intermediate (often produced from a neighboring promoter).

    • Examples: F-element (Drosophila), LINE & SINE elements (Homo sapiens).

Example of Adaptive Evolution via Transposons: Industrial Melanism in Peppered Moths

  • The replacement of the common pale typica form of the peppered moth (Biston betularia) by the black carbonaria form during the Industrial Revolution is a classic example of visible evolutionary response to environmental change (bird predation and coal pollution).

  • The mutation causing industrial melanism in British peppered moths is due to the insertion of a large, tandemly repeated, transposable element (a Class 2 DNA type, 2.5 x ~9 kb repeat) into the first intron of the cortex gene.

  • This insertion upregulates the expression of cortex, resulting in increased darker coloration. The cortex gene is known to control mimicry and crypsis in other moths and butterflies.

Integrons 🧩

  • Integrons are genetic entities capable of capturing small mobile elements called gene cassettes.

  • All integrons contain three key elements:

    1. intI: A gene encoding an integrase of the tyrosine recombinase family.

    2. attI: A primary recombination site for cassette insertion.

    3. Pc: An outward-oriented promoter that drives the expression of captured gene cassettes.

  • Structure:

    • Stable platform: Contains intI (recombinase gene), PintI (its promoter), and attI (primary recombination site).

    • Variable part: Formed by an array of gene cassettes, each typically containing a single gene (ORF) and an attC recombination site (also known as a 59-base element).

  • Gene Acquisition:

    • The IntI integrase mediates site-specific recombination between the attI site on the integron and an attC site on a circular gene cassette. This integrates the gene cassette downstream of the Pc promoter, allowing for gene expression.

    • Additional cassettes can be integrated (or excised) similarly.

  • Gene Rearrangement:

    • The IntI integrase can also mediate the excision of any gene cassette within the integron array by recombination between two attC sites surrounding the gene.

    • The excised cassette can then be reinserted at the attI site, often placing it closer to the Pc promoter and thus potentially increasing its expression level. Gene cassettes located too far from Pc may not be expressed efficiently.

HGT in the Tree of Life 🌳

  • Endosymbiosis and Symbiogenesis: Major HGT events in eukaryotic evolution include:

    • Mitochondria derived from Proteobacteria.

    • Plastids (e.g., chloroplasts) derived from Cyanobacteria.

  • Unexpected Phylogenetic Distributions: HGT can lead to gene phylogenies that do not match the organismal phylogeny. If organisms A, B, C, and D have a known evolutionary relationship (e.g., (A,(B,(C,D)))), but a specific gene variant is shared by A and C but different in B and D, HGT is a possible explanation.

  • Example: Pea Aphids and Carotenoid Biosynthesis:

    • Phylogenetic analyses of genes encoding enzymes for carotenoid biosynthesis (e.g., carotenoid cyclase–carotenoid synthase, carotenoid desaturase) in pea aphids show that these genes are closely related to fungal homologues, not to those of other animals. This suggests aphids acquired these genes via HGT from fungi.

  • A Reticulated Tree of Life: Due to the prevalence of HGT, especially in prokaryotes but also impacting eukaryotes, the history of life may be better represented by a reticulated tree (a net) rather than a simple branching tree.

Learning Objectives Review

This lecture covered:

  • Different types of transposons and their examples.

  • The two main models of transposition (replicative and non-replicative).

  • Genetic rearrangements produced by transposons.

  • The structure and function of integrons in capturing and expressing gene cassettes.