Development of the Micromonospora carbonacea var. africana ATCC 39149 bacteriophage pMLP1 integrase for site-specific integration in Micromonospora spp.
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 tRNAHis 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.
Alexander
Dylan C
DC
New Lead Discovery, Schering Plough Research Institute, 2015 Galloping Hill Road, K15-B425-MS4800, Kenilworth, NJ 07033, USA.
Devlin
David J
DJ
New Lead Discovery, Schering Plough Research Institute, 2015 Galloping Hill Road, K15-B425-MS4800, Kenilworth, NJ 07033, USA.
Hewitt
Duane D
DD
New Lead Discovery, Schering Plough Research Institute, 2015 Galloping Hill Road, K15-B425-MS4800, Kenilworth, NJ 07033, USA.
Horan
Ann C
AC
New Lead Discovery, Schering Plough Research Institute, 2015 Galloping Hill Road, K15-B425-MS4800, Kenilworth, NJ 07033, USA.
Hosted
Thomas J
TJ
New Lead Discovery, Schering Plough Research Institute, 2015 Galloping Hill Road, K15-B425-MS4800, Kenilworth, NJ 07033, USA.
D001287:Attachment Sites, Microbiological / Q000235:genetics; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D004269:DNA, Bacterial / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli; D005798:Genes, Bacterial; D005822:Genetic Vectors; D015698:Genomic Library; D019426:Integrases / Q000235:genetics*; D008847:Micromonospora / Q000145:classification / Q000235:genetics* / Q000821:virology*; D008969:Molecular Sequence Data; D010957:Plasmids; D012354:RNA, Transfer, His / Q000737:chemistry* / Q000235:genetics; D011995:Recombination, Genetic; D016662:Virus Integration / Q000235:genetics
D004269:DNA, Bacterial; D004279:DNA, Viral; D012354:RNA, Transfer, His; D019426:Integrases