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

Dylan C. Alexander

New Lead Discovery, Schering Plough Research Institute, 2015 Galloping Hill Road, K15-B425-MS4800, Kenilworth, NJ 07033, USA

Correspondence
Thomas Hosted
thomas.hosted{at}spcorp.com

Micromonospora carbonacea var. africana ATCC 39149 contains a temperate bacteriophage, pMLP1, that is present both as a replicative element and integrated into the chromosome. Sequence analysis of a 4·4 kb KpnI fragment revealed pMLP1 att/int functions consisting of an integrase, an excisionase and the phage attachment site (attP). Plasmids pSPRH840 and pSPRH910, containing the pMLP1 att/int region, were introduced into Micromonospora spp. by conjugation from Escherichia coli. Sequence analysis of DNA flanking the integration site confirmed site-specific integration into a tRNA^His gene in the chromosome. The pMLP1 attP element and chromosomal bacterial attachment (attB) site contain a 24 bp region of sequence identity located at the 3' end of the tRNA. Integration of pMLP1-based plasmids in M. carbonacea var. africana caused a loss of the pMLP1 phage. Placement of an additional attB site into the chromosome allowed integration of pSPRH840 into the alternate attB site. Plasmids containing the site-specific att/int functions of pMLP1 can be used to integrate genes into the chromosome.

The GenBank accession numbers for the sequences reported in this paper are AY150027 (M. carbonacea var. africana pMLP1 att/int region), AY150029, AY150033 and AY150028 (M. carbonacea var. africana pSPRH840 attL, attR and attB sites, respectively), and AY150031, AY150032 and AY150030 (M. halophytica var. nigra pSPRH840 attL, attR and attB sites, respectively).

Present address: Cubist Pharmaceuticals, 65 Hayden Avenue, Lexington, MA 02421, USA.

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