Doctor of Philosophy (PhD)
For Streptomyces plasmid pIJ101 to mediate DNA transfer, only the clt locus and the Tra protein are required, though three non-essential spread functions increase the radial distance of plasmid dissemination among recipients after the initial lateral transfer event. clt is a contiguous 54-bp sequence within an intrinsically curved plasmid region that contains inverted and direct repeats; biochemical and genetic methods described here indicate that single-stranded cleavage reminiscent of most conjugative plasmids does not occur at clt. clt therefore likely promotes plasmid transfer by interacting with the pIJ101 Tra protein or host-encoded factors in a transient, novel DNA-processing event or by mediating transfer of unprocessed, covalently closed circular plasmids. Tra promotes plasmid and chromosome mobilization by an unknown mechanism and shows similarity to ATP-binding proteins that mobilize double-stranded DNA. Here, epitope sequences were inserted at two positions within Tra, one dispensable for conjugation, and one previously found to be important for chromosome mobilization; these epitopes allowed for identification of Tra in Western blots of E. coli and Streptomyces, as well as identification of the protein in Streptomyces by immunofluorescence microscopy. The pIJ101 kilB spread gene is lethal unless the plasmid-encoded kil-override gene, korB, is also present. The 10-kDa KorB protein is processed to a 6-kDa repressor, which has previously been solely detected in S. lividans. S. cyanogenus plasmid pSB24.1 undergoes spontaneous deletion in S. lividans to form the stable, non-conjugative pSB24.2, which is similar in sequence and organization to a portion of pIJ101, including analogous korB and clt functions and the 3' end of a putative kilB gene. The korB gene from pSB24.2 previously overrode pIJ101 kilB-associated lethality, and here, KorB (pSB24.2) repressed kilB promoter transcription as efficiently as KorB (pIJ101). Western blotting demonstrated that KorB of pSB24.2 exists in both S. lividans and S. cyanogenus in several forms ranging from 10 to 6 kDa. KorB from pIJ101 meanwhile persists in only its 6-kDa form in both species, and increased expression of both KorB proteins is strongly correlated to the sporulation phase of the streptomycete life cycle.
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Ducote, Matthew John, "Transfer functions of Streptomyces plasmid pIJ101: insights into a novel bacterial conjugation system" (2004). LSU Doctoral Dissertations. 246.