Development of the Micromonospora carbonacea var. africana ATCC 39149 bacteriophage pMLP1 integrase for site-specific integration in Micromonospora spp

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• The temperate bacteriophage pMLP1 resides in Micromonospora carbonacea var. africana ATCC 39149, existing as both a free element and integrated into the host chromosome.
• Understanding bacteriophage integration mechanisms is crucial for genetic manipulation of bacterial hosts.

Purpose of the Study:

• To characterize the att/int functions of bacteriophage pMLP1.
• To investigate the site-specific integration mechanism of pMLP1 into the Micromonospora chromosome.
• To assess the potential of pMLP1 integration system for gene delivery into Micromonospora species.

Main Methods:

• Sequence analysis of a 4.4 kb KpnI fragment of pMLP1 to identify att/int genes.
• Construction of plasmids (pSPRH840, pSPRH910) containing the pMLP1 att/int region.
• Introduction of plasmids into Micromonospora spp. via conjugation from Escherichia coli.
• Analysis of DNA flanking integration sites to confirm chromosomal integration.

Main Results:

• The pMLP1 att/int region encodes an integrase, an excisionase, and the phage attachment site (attP).
• Site-specific integration occurred within a tRNAHis gene in the Micromonospora chromosome, involving a 24 bp sequence identity between attP and the chromosomal attB site.
• Integration of pMLP1-based plasmids into M. carbonacea resulted in the loss of the pMLP1 phage.
• An additional attB site enabled integration of a plasmid into an alternative chromosomal location.

Conclusions:

• The pMLP1 integrase and attachment sites mediate site-specific integration into the Micromonospora chromosome.
• The pMLP1 integration system offers a tool for targeted gene integration into Micromonospora species.
• This system has potential applications in genetic engineering of Micromonospora for biotechnological purposes.