3) An analysis of the sequence space between the repA and parA g

3). An analysis of the sequence space between the repA and parA genes of plasmids pISP0, pLA1, pSLGP and pSPHCH01

(165–195 bp; see Table 1) did not identify any significant repeated sequences. Thus, it can be concluded that the organization of the rep and par genes on the ‘megaplasmids’ Compound C from sphingomonads belonging to the ‘Mega-RPA-’ and ‘Mega-Rep3-’ groups differs significantly from those previously described for plasmids from other Alphaproteobacteria by Petersen (2011). There are only very few studies available, which analysed the transferability of the ‘degradative megaplasmids’ from sphingomonads. In these studies, it was shown for plasmids pNL1 and pCHQ1 (inter alia using plasmid derivatives carrying antibiotic resistance markers) that the transfer (or the ability to establish in a different genetic background) of these plasmids seems to be basically restricted

to bacteria belonging to the Sphingomonadaceae (Romine et al., 1999; Basta et al., 2004, 2005; Nagata et al., 2006). The conjugative systems of Gram-negative bacteria show in general three essential components: a type IV secretion system which spans the cell envelope and is responsible for the synthesis of the conjugative pili; the relaxosome which is a complex of proteins PLX4032 cost that process the DNA at the origin of transfer (oriT) OSBPL9 and the coupling protein, which connects the two entities together (Lawley et al., 2004). The type IV secretion systems are rather complex and usually require more than 10 different proteins, which are involved in functions such as the synthesis of the pili and the formation of pores through the inner and outer membranes and the cell walls of the donor and

recipient cells. Historically, the proteins/genes involved have been designated differently for different plasmids (especially when these plasmids belong to different incompatibility groups). Thus, the relevant proteins have been designated as Tra(X) for plasmids belonging to the incompatibility groups IncF1 and IncN, as Trb(X) for plasmids from the IncPα group, Trw(X) for IncW plasmids or VirB(1–10) for Ti plasmids (Lawley et al., 2004). Therefore, the sequences of the sphingomonad plasmids were analysed for the presence of annotated tra, trh, trb, trw or vir genes. This demonstrated that only in plasmids with sizes of c. 50–310 kbp gene clusters with 10 or more genes annotated as tra, trb, trw or vir are present. Plasmids pNL1 and pCAR3 (from the ‘Mega-RepAC group’) carried the genes required for conjugative transfer on parts of the plasmids with a length of about 20 kb. These genes have been annotated for pNL1 and pCAR3 mostly as tra genes (traL, traE, traK, traB, traC, traW, traU, traN, traF, traH, traG, traI, traH).

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