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Integration and excision of the Mycobacterium tuberculosis prophage-like element, phiRv1
Lori A Bibb1, Graham F Hatfull
1Department of Biological Sciences, University of Pittsburgh, PA 15260, USA.
Abstract:
The genomes of Mycobacterium tuberculosis H37Rv and CDC1551 each contain two prophage-like elements, phiRv1 and phiRv2. The phiRv1 element is not only absent from Mycobacterium bovis BCG but is in different locations within the two sequenced M. tuberculosis genomes; in both cases phiRv1 is inserted into a REP13E12 repeated sequence, which presumably contains the bacterial attachment site, attB, for phiRv1. Although phiRv1 is probably too small to encode infectious phage particles, it may nevertheless have an active integration/excision system and be capable of moving from one chromosomal position to another. We show here that the M. tuberculosis H37Rv phiRv1 element does indeed encode an active site-specific recombination system in which an integrase of the serine recombinase family (Rv1586c) catalyses integration and excision and a small, basic phiRv1-encoded protein (Rv1584c) controls the directionality of re-combination. Integration-proficient plasmid vectors derived from phiRv1 efficiently transform BCG, can utilize four of the seven REP13E12 sites present in BCG as attachment sites, and can occupy more than one site simultaneously.
Insights
Mycobacterium tuberculosis harbors mobile genetic elements like phiRv1. This study reveals phiRv1 encodes a site-specific recombination system enabling its movement within the genome and facilitating genetic transfer in Mycobacterium bovis BCG.
Area of Science:
- Microbiology
- Genomics
- Molecular Biology
Background:
- Mycobacterium tuberculosis genomes contain prophage-like elements, including phiRv1 and phiRv2.
- The phiRv1 element exhibits variability in location across M. tuberculosis strains and is absent in Mycobacterium bovis BCG.
- phiRv1 is inserted into REP13E12 repeat sequences, suggesting these contain the bacterial attachment site (attB).
Purpose of the Study:
- To investigate the functional capabilities of the phiRv1 element from Mycobacterium tuberculosis H37Rv.
- To determine if phiRv1 possesses an active integration/excision system and can mediate chromosomal mobility.
- To explore the potential of phiRv1-derived vectors for genetic manipulation in mycobacteria.
Main Methods:
- Analysis of phiRv1 sequences and genomic locations in M. tuberculosis H37Rv and CDC1551.
- Identification and characterization of genes encoding the recombination system (integrase Rv1586c and regulatory protein Rv1584c).
- Construction and testing of integration-proficient plasmid vectors derived from phiRv1 for transformation of Mycobacterium bovis BCG.
Main Results:
- The phiRv1 element encodes an active site-specific recombination system, including a serine recombinase family integrase (Rv1586c) and a directionality-controlling protein (Rv1584c).
- phiRv1-derived plasmid vectors efficiently transform Mycobacterium bovis BCG.
- These vectors can utilize multiple REP13E12 sites in BCG as attachment sites, with some vectors occupying more than one site simultaneously.
Conclusions:
- The phiRv1 element possesses a functional site-specific recombination system enabling its mobility and potential for horizontal gene transfer.
- phiRv1-derived vectors represent a promising tool for genetic engineering and manipulation of Mycobacterium bovis BCG.
- Understanding phiRv1's mobility is crucial for comprehending mycobacterial genome dynamics and developing novel genetic tools.