MICR 321: Advanced Microbiology - Lecture 10: Transposition & Transposon Mutagenesis

MICR 321: Advanced Microbiology

Transposons: Also referred to as "jumping genes", they are DNA elements capable of hopping or transposing from one position in the DNA to another.
- Historical Context: Discovered by Barbara McClintock in corn during the early 1950s.
- Existence: Present in every organism on Earth; it is estimated that approximately half of the human genome consists of transposons.

Transposition: The process through which transposons move within the genome.
Transposases: Enzymes that facilitate transposition; these are encoded within the transposon itself.
Importance of Transposition: Provides a mechanism for transferring genes from one bacterium to another, even when there is little sequence homology.
Recombination Types:
- Homologous Recombination: Predominantly involves DNA molecules with similar or identical sequences; this method utilizes "homology arms" during cloning.
- Non-Homologous Recombination: Does not require sequence similarity, relying instead on enzymes that recognize specific DNA regions regardless of sequence similarity.

Result: The transposon is relocated to a different genomic site than its original location.
Process:
- DNA is cut from the original strand (donor DNA) and may or may not be copied before being inserted into a different location (recipient DNA).
- The transposase enzyme plays a crucial role by cutting the donor DNA at the transposon's ends and inserting it into the target DNA.
- Terminology:
- Donor DNA: The original DNA strand where the transposon is excised.
- Recipient DNA: The DNA strand into which the transposon inserts.
Regulation of Transposition: It is critical for transposition to be tightly regulated to maintain genomic stability, as uncontrolled transposition could lead to detrimental genomic changes.

Size and Composition:
- Smaller Transposons: Approximately 1000 bp; encode only the transposase gene.
- Larger Transposons: Include additional regulatory genes or genes that confer advantages such as antibiotic resistance (e.g., Tet resistance).
Common Features:
- Inverted Repeats: Found at the ends of bacterial transposons and recognized by transposases, which facilitate excision and integration.
- Direct Repeats: Generated in the target DNA post-integration, flanking the inserted transposon.

Insertion Sequence (IS) Elements:
- Smallest bacterial transposons (about 750 to 2000 bp); do not carry selectable genes.
- Typically inactivate genes into which they insert, mimicking deletion mutations but can revert.
- Polar Effects: Transposition can affect downstream gene expression.
Composite Transposons:
- Formed when two copies of the same IS elements join together to create a larger transposon that can transfer flanking regions along with the genes contained between them (often encodes selectable genes).

IS Element Structure:
- Contains only a transposase gene.
Composite Transposon Structure:
- Comprising two IS elements plus additional genes (e.g., gene A).

Reverse Genetics: A technique that involves targeting and creating a mutation in a specific gene locus to observe the effects on phenotype and function.
Forward Genetics: Sequence begins with a phenotype/function to identify the associated gene locus.

Qualities of Effective Mutagenic Transposons:
- High Frequency: Should transpose at a high rate.
- Non-specific Targeting: Should not be highly selective for a specific DNA sequence.
- Selectable Markers: Must carry easily identifiable selectable markers (such as antibiotic resistance genes).
- Broad Host Range: Capable of transposing across diverse bacterial species.
Mutant Cloning: Selected transposons can be cloned into vectors that do not replicate inside the host; this allows transposition from the plasmid to the genome, disrupting targeted genes.
High-throughput Methods: This approach allows for the rapid generation of mutations, even in species without a complete genome sequence, leveraging newer integration techniques with next-generation sequencing technologies.

Functionality Impact: Insertions in coding regions, particularly within the first half of a gene, typically lead to loss-of-function alleles.
Coverage Strategy: Ideally, one wants a single insertion per gene, ensuring extensive coverage across as many genes in the genome as possible. Using insights from previous collections (like the Keio collection) helps in strategic planning for gene coverage.
Mapping Mutations: Traditional methods involved cloning or arbitrary PCR following selection, whereas newer approaches utilize TnSeq coupled with next-generation sequencing to allow concurrent mapping of mutations and streamlined analysis post-screening.
Barcoded Transposon Libraries: A novel method described by Wetmore et al. (2015) enables high-throughput screening of mutations and their functional annotations using improved barcoding techniques.

Create Transposon Insertion Site (TIS) Library:
- Introduce random mutations and isolate the genomic DNA.
- Select, pool mutants, and fragment the DNA, subsequently adding adaptors and PCR amplifying.
Sequencing and Mapping: Extracted DNA is subjected to sequencing and mapping to identify transposon insertion locations across the genome.

Saturated Krmit-Tn Library:
- A modified mariner transposon inserting at TA sites, achieving 68,857 unique insertion sites with an average insertion every ~35 nt.
Library Pooling vs. Arraying: When stratifying mutants for analysis, both pooling and arraying confer unique advantages and disadvantages, impacting the experimental approach.

Quality Control: Reads checked and trimmed, mapped to reference genomes to verify insertion sites and discard irrelevant reads.
Differential Analysis: Comparing input mutant pools against output to determine underrepresentation which implies reduced fitness or survival of specific mutants under selective conditions.
Applications of Data: This data leads to insights about bacterial fitness in various conditions, underscoring the relevance of the results in experimental microbiology.

Development: Allows rapid fitness profiling and function annotation using random barcodes and TnSeq—integrates seamlessly in modern microbial work, significantly increasing throughput and simplifying complex protocols.