Characterization of a Rap-Phr-like system and a conjugation-associated ORF of pAW63 from Bacillus thuringiensis

Conjugation is an efficient mechanism of Horizontal Gene Transfer used by bacteria to adapt to changing environment and to evolve. This mechanism has been extensively studied in Gram-negative bacteria where it has been shown that the mobile DNA is processed and transported through a Type IV Secretion System (T4SS). Recently, a homologous system was found in the Gram-positive plasmid pIP501 from Streptococcus agalactiae, and has been called Type IV Secretion-Like System (T4SLS). However, the question of partner recruitment in Gram-positive conjugation and T4SLS regulation remained mainly unanswered. pAW63 from B. thuringiensis sv. kurstaki HD73 is the model conjugative plasmid from the B. cereus s.l. group. During this work, its sequence has been updated through bioinformatics analysis and led to the discovery of three plasmids (or plasmid-like molecules) homologous to pAW63, increasing the family of pXO2-like plasmids to six members. New putative functions have also been postulated for three ORFs. Protein encoded by pAW63 orf17 was found to be homologous to pilin from Gram-negative sex pili while the ones from ORFs 67 ad 70 could be accessory proteins of the relaxosome complex. These homologies should be tested experimentally but bring new insight into the T4SLS apparatus of pAW63. Focus was then brought on the biological role of the pAW63-encoded RapD-PhrD putative quorum sensing system and of the orf26. The phrD was found to be actively transcribed from transition to stationary phase. The involvement of the RapD-PhrD system in regulation of pAW63 conjugative transfer and in sporulation was tested. There was no conclusive proof regarding the involvement of this system in conjugation whereas sporulation seemed likely to be its biological function. Regarding orf26, it is the largest of pAW63 and knock-out mutation showed that it abolished self-transfer ability of pAW63. These results should be confirmed but definitely pave the way for further characterization of pAW63 T4SLS system and regulation.(AGRO 3) -- UCL, 201