Chromosomal DNA transfer in Mycobacterium smegmatis is mechanistically different from classical Hfr chromosomal DNA transfer

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Abstract
Classical conjugal DNA transfer of chromosomal DNA in bacteria requires the presence of a cis-acting site, oriT, in the chromosome. Acquisition of an oriT occurs if a conjugative plasmid integrates into the chromosome to form an Hfr donor strain, which can transfer extensive regions of chromosomal DNA. Because oriT sequences are unique, and because transfer occurs in a 5′ to 3′ direction, the frequency with which a particular gene is inherited by the recipient depends on the gene's location: those closest to the 3′ side of oriT are transferred most efficiently. In addition, as the entire chromosome must be transferred to regenerate oriT, Hfr transconjugants never become donors. Here we describe novel aspects of a chromosomal DNA transfer system in Mycobacterium smegmatis. We demonstrate that there are multiple transfer initiations from a donor chromosome and, as a result, the inheritance of any gene is location-independent. Transfer is not contiguous; instead, multiple non-linked segments of DNA can be inherited in a recipient. However, we show that, with appropriate selection, segments of DNA at least 266 kb in length can be transferred. In further contrast to Hfr transfer, transconjugants can become donors, suggesting that the recipient chromosome contains multiple cis-acting sequences required for transfer, but lacks the trans-acting transfer functions. We exploit these observations to map a donor-determining locus in the M. smegmatis chromosome using genetic linkage analysis. Together, these studies further underline the unique nature of the M. smegmatis chromosomal transfer system.