Open AccessMycobacteriophage D29 contains an integration system similar to that of the temperate mycobacteriophage L5
Author(s) -
Graça Ribeiro,
Miguel Viveiros,
Hugo L. David,
João Costa
Publication year - 1997
Publication title -
microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.352
H-Index - 35
eISSN - 1465-2080
pISSN - 1350-0872
DOI - 10.1099/00221287-143-8-2701
Subject(s) - integrase , biology , genetics , plasmid , mycobacterium smegmatis , gene , temperateness , nucleic acid sequence , microbiology and biotechnology , intergenic region , transposable element , dna , bacteriophage , escherichia coli , genome , mycobacterium tuberculosis , medicine , tuberculosis , pathology
Summary: A mycobacteriophage D29 DNA fragment cloned in pRM64, a shuttle plasmid that transforms Mycobacterium smegmatis , was sequenced. The determined sequence was 2592 nucleotides long and had a mean G+C content of 63.7 mol%, similar to that of mycobacterial DNA. Four ORFs were identified: one with strong homology to dCMP deaminase genes; one homologous to mycobacteriophage L5 gene 36, whose function is unknown; one encoding a possible excisase; and one encoding an integrase. The intergenic region between the putative excisase gene and the integrase gene had a lower than average G+C content and showed the presence of the same attP core sequence as mycobacteriophage L5. Transformation experiments using subclones of pRM64 indicated that the integrase gene and all the intergenic region were essential for stable transformation. A subclone containing the integrase gene and the core attP sequence was able to transform but recombinants were highly unstable. Southern analysis of total DNA from cells transformed with pRM64 and its derivatives showed that all the plasmids were integrated at one specific site of the bacterial chromosome. A recombinant exhibiting a high level of resistance to the selective drug kanamycin had two plasmids integrated at different sites. These results demonstrated that the D29 sequences contained in pRM64 were integrative, indicating that the generally held view of D29 as a virulent phage must be reviewed.