Conjugation of extrachromosomal replicons of Rhodococcus erythropolis AN12

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2006.Includes bibliographical references.Bacteria belonging to the Gram-positive actinomycete species, Rhodococcus erythropolis, are diverse not only in terms of metabolic potentials but the plasmids they encode. Pulsed-field gel electrophoresis (PFGE) revealed three previously uncharacterized megaplasmids in the genome of Rhodococcus erythropolis AN12. These megaplasmids, pREA400, pREA250 and pREA100, migrate at approximately 400 kb, 250 kb and 100 kb, respectively. Genetic screening of an AN12 transposon insertion library showed that two megaplasmids, pREA400 and pREA250, are conjugative. It is known for other bacterial systems that a relaxase encoded by the traA gene is required to initiate DNA transfer during plasmid conjugation. Sequences adjacent to the transposon insertion in megaplasmid pREA400 revealed a putative traA-like open reading frame. A novel site-specific gene disruption method was developed to generate a traA mutation in AN12, which allowed us to address the role of the traA gene for Rhodococcus megaplasmid conjugation. We found that the AN12 traA mutant is no longer capable of transferring the pREA400 megaplasmid to Rhodococcus erythropolis SQ1. It was shown previously that the R. erythropolis AN12 genome harbors a 6.3 kb cryptic plasmid called pAN12.(cont.) Here we show that pAN12 is conjugatively mobilizable into other rhodococcal strains. A series of plasmid deletion constructs were tested for loss of mobility to identify the pAN12 cis-acting conjugation requirement. In this way, an approximately 700 bp region was found to be required for plasmid transmission. A small 61 bp element within this region exhibited sequence similarity to the minimal 54 bp clt region known to be required for the conjugation of the streptomycete plasmid, pIJ101. The functionality of these cis-acting elements appears to be conserved, as the addition of this pAN12 clt-like region confers mobility to an otherwise non-conjugative plasmid. However, unlike pJI 101 which encodes all necessary factors for transfer, pAN12 mobility is dependent on the presence of the AN12 megaplasmid, pREA400.by Joyce Chun-Yi Yang.Ph.D