Lecture 15 - Horizontal Gene Transfer

Question 1

What is horizontal gene transfer?

Answer 1

‘Transfer of genes between organisms by means other than reproduction’.

The weight of evidence still supports the likelihood that mitochondria in eukaryotes derived from alpha-proteobacterial cells and that chloroplasts came from ingested cyanobacteria

Major mechanism for the spread of antibiotic resistance
Responsible for transfer of virulence traits
‘Quantum leaps’ of evolution over a short period of time

Laterally transferred DNA comprises:

• Phage
• Plasmids
• Smaller genomic islets (< 10 kb)
• Genomic islands (GEIs), >10 kb
• “Simple” and conjugative transposons

How does the DNA integrate into its new host?

Homologous recombination
Site specific recombination
Transposition

Question 2

Insertion Sequences

Answer 2

type 1: Insertion sequences/integrons

IS’s have inverted repeats (about 10 to 40 bp) at their termini (IR)

Upon insertion into chromosome the target site is duplicated (FDRs/DRs - flanking direct repeats)

The sequences of the inverted repeats at each end of the IS are very similar but not necessarily identical.

Vey simple, up to about 2 kb in length, only encode transposase

Transposase facilitates the behaviour of the insertion sequence jumping (restriction endonuclease part to excise and insert itself elsewhere)

Question 3

Transposons class 1 - COMPOSITE

Answer 3

https: //www.youtube.com/watch?v=ul8BmgqN2YY
https: //www.youtube.com/watch?v=eSD_tbjfSlA

• Class I elements (composite transposons)

Composite transposons have two or more insertion sequences with DNA inbetween (always have to have at least one transposase encoded for).
Because composite transposons have two or more IRs the DNA that ‘jumps’ around can contain different DNA sequences - generating new transposons.
Depending on where it inserts itself, when it excises itself again it can take new segments of DNA with it.

NO intermediate.
Encode a multifunctional enzyme called transposase.
Transposase recognises the DNA sequences flanking the transposon. It cuts the transposon out of the DNA and reinserts it elsewhere - cut and paste mechanism of transposition.

Question 4

Transposons class 2 - REPLICATIVE

Answer 4

• Class II elements (Replicative (Tn 3 family) transposons)

Are transcribed into an DNA intermediate.
This is copied back into DNA by reverse transcriptase (encoded by the transposon)
DNA is then re-integrated into the genome.
(the original transposon never leaves the DNA).

Resolvase enzyme activity is involved in site-specific recombination between two transposons present as direct repeats in a cointegrate structure

Question 5

Anatomy of a transposon

Answer 5

Encodes a gene for transposase flanked at either end by terminal Inverted Repeats (sequences that are reverse compliments of each other)
The Inverted Repeats serve as the transposase binding and cutting site
Either end of the IR are flanked by DRs - Direct Repeats (a result of the insertion of the transposon into the DNA). NOT part of the transposon and are left behind when the transposon leaves the DNA.

Question 6

Steps of transposition

Answer 6

1. Excision
2. Drift
3. Integration
4. EXCISION
5. Transposase is transcibed and translated from the transposon.
6. Transposase binds to the terminal inverted repeats, dimerises (‘transposome’), and cleaves the transposon from the donor site
7. DRIFT
8. Transposase carries the transposon to the target site
9. INTEGRATION
10. Recipient DNA is cleaved by transposase and the transposon inserted
11. DNA ligase integrates transposon into the genome

The DNA donor site contains evidence of the transposon through the direct repeat duplication in the target site.

The DNA recipient site contains a new target site duplication that resulted from the insertion

Question 7

Conjugative transposons

Answer 7

Important in antibacterial resistance in gram positives.

Conjugative transposons have a closed circular transposition intermediate (plasmid like)

Do not duplicate the target site when they integrate into DNA.

Intracellular transposition: Are plasmid-like, transferred by conjugation, but the circular intermediate of a conjugative transposon does not replicate

Conjugative transposon is excised, and either enters the plasmid or back into the genome.

Intercellular transposition: Plasmid is excised, transferred to neighbouring cell, and pseudo replicated and then integrated.

Question 8

Horizontal Gene Transfer

Answer 8

1. Transformation
   Transduction
   Conjugation

Question 9

1. Transformation

Answer 9

Natural transformation

Competence - naturally uptake bacteria

Uptake - Conversion to single stranded DNA
Stabilisation

Integration - Homologous recombination

Competency
SSBPs facilitate unwinding to ssDNA
Occurs at exponential growth
Expression of 8-10 proteins
Several pathogens have natural competency
Acinetobacter and PA
Important genetic exchange mechanism in the soil, aquatic ecosystems and biofilms
Competency proteins and secretins in G- uptake DNA (also used in secretion of proteins in a TypeIISS!!)
There is a DNA uptake signal receptor that causes PilQ to pull it in and unwinds it, transfers it through Com with SSBPs

H. Pylori:
use a type 4 secretion system for pulling in DNA

Summary:
In competent cells, you have conserved proteins (competency proteins) in the inner membrane. In g+ there are competency proteins in the inner membrane that have homology with T2SS. In H. Pylori, T4SS is used.

Question 10

Conjugation

Answer 10

Cell to cell contact
Plasmid encoded mechanism
F plasmid (fertility)

They get across the membrane through a T4SS - fundamental to transfer of DNA OUT of cell during conjugation. Also for uptake in helicobater (rare)

Nanotubes: B. subtilis can transfer GFP molecules from one population to another over time

Question 11

Why isn’t HGT more widespread?

Answer 11

RM systems: Restriction modification systems - defence systems

Restriction enzymes cleave foreign DNA, it protects it’s own DNA sequences that are the same by methylating the DNA

This is a massive barrier to random DNA uptake.

Crispr-cas works like this.

Question 12

Examples of HGT in pathogens

Answer 12

C. difficile

Genome of strain 630 contains at least 7 conjugative transposons
Ribotype 027 is lethal as contains resistance to every antibiotic
New 078 ribotype contains a novel 100kb genomic island that contains a novel transposon Tn6164
Tn6164 has over 90 ORF’s including several encoding for antibiotic resistance

MRSA

Schematic representation of the staphylococcal cassette chromosome mec (SCCmec). Example of a genomic island
CcrA and CcrB important in mobilisation

Range from 20-70kb

Can also be transferred by bacteriophage