Cloning of Frankia species putative tRNA(Pro) genes and their efficacy for pSAM2 site-specific integration in Streptomyces lividans.
pSAM2 is a conjugative Streptomyces ambofaciens mobile genetic element that can transfer and integrate site specifically in the genome. The chromosomal attachment site (attB) for pSAM2 site-specific recombination for two Frankia species was analyzed. It overlaps putative proline tRNA genes having a 3'-terminal CCA sequence, an uncommon feature among actinomycetes. pSAM2 is able to integrate into a cloned Frankia attB site harbored in Streptomyces lividans. The integration event removes the 3'-terminal CCA sequence and introduces a single nucleotide difference in the T psi C loop of the putative Frankia tRNA(Pro) gene. Major differences between the attP sequence from pSAM2 and the Frankia attB sequence restrict the identity segment to a 43-bp-long region. Only one mismatch is found between these well-conserved att segments. This nucleotide substitution makes a BstBI recognition site in Frankia attB and was used to localize the recombination site in a 25-bp region going from the anticodon to the T psi C loop of the tRNA(Pro) sequence. Integration of pSAM2 into the Frankia attB site is the first step toward introduction of pSAM2 derivatives into Frankia spp.
Alegre
M T
MT
Laboratoire de Microbiologie des Sols, URA CNRS 1450, Université Claude Bernard-Lyon I, Villeurbanne, France.
Cournoyer
B
B
Mesas
J M
JM
Guérineau
M
M
Normand
P
P
Pernodet
J L
JL
D000191:Actinomycetaceae / Q000235:genetics*; D001287:Attachment Sites, Microbiological / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012361:RNA, Transfer, Pro / Q000737:chemistry / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000235:genetics*
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D012361:RNA, Transfer, Pro