Is Closely Related to Linear Plasmids of Other Rhodococcus Strains

Functional analysis of the linear replicon pFiD188 has shown that it carries covalently attached proteins on the 5'-terminal ends. However, no extensive TIRs, typical for invertrons, could be detected in pFiD188 (unpublished data). This lack of long TIRs is a common characteristic of several rhodococcal linear plasmids (Kalkus et al. 1998; Stecker et al. 2003; Sekine et al. 2006).

Homology searches with the available sequences reveal that pFiD188 has no homology to the circular virulence plasmid of R. equi, but has a limited conservation with genes on linear plasmids of other actinomycetes, such as SCP1 of S. coelicolor A3(2) (Bentley et al. 2004), plasmid 1 of Mycobacterium species MCS (accession CP000385), and pRHL1 and pRHL3 of Rhodococcus sp. strain RHA1 (Warren et al. 2004; McLeod et al. 2006). However, pFiD188 is strikingly similar to the linear plasmids pBD2 and pREL1 of R. erythropo-lis strains BD2 and PR4 (Stecker et al. 2003; Sekine et al. 2006), and pRHL2 of Rhodococcus sp. strain RHA1 (Shimizu et al. 2001; McLeod et al. 2006; Fetzner et al., in this volume), suggesting a common origin (Fig. 2). The two R. ery-thropolis plasmids exhibit six regions of extensive homology (R1 through R6; Sekine et al. 2006). After comparative analysis of the four linear replicons with the Artemis comparison tool (Carver et al. 2005), four colinear regions (labeled R1, R2, R3, and R6) and three regions unique for each plasmid (labeled U1, U2, and U3) were found. The conserved regions probably code for plas-mid replication, maintenance, and partitioning functions. However, because

Fig. 2 Sequence comparison of the linear plasmids. The Artemis comparison tool (Carver et al. 2005) was used to plot protein similarities (tblastx) between pFiD188 (R. fas-cians), pBD2 and pRELl (R. erythropolis strain BD2 and PR4, respectively), and pRHL2 (Rhodococcus sp. strain RHA1). Lines drawn between two adjacent linearized replicons show the location of homologous genes; darker lines represent higher-quality matches. Conserved (R) and unique (U) regions are indicated. The black bar flanking region R6 in pFiD188 depicts the site of the sequence gap

Fig. 2 Sequence comparison of the linear plasmids. The Artemis comparison tool (Carver et al. 2005) was used to plot protein similarities (tblastx) between pFiD188 (R. fas-cians), pBD2 and pRELl (R. erythropolis strain BD2 and PR4, respectively), and pRHL2 (Rhodococcus sp. strain RHA1). Lines drawn between two adjacent linearized replicons show the location of homologous genes; darker lines represent higher-quality matches. Conserved (R) and unique (U) regions are indicated. The black bar flanking region R6 in pFiD188 depicts the site of the sequence gap the regions R2, R3, and R6 mostly hold genes that encode hypothetical proteins, it is impossible to predict their role in these processes. On the other hand, region R1 comprises putative genes for the telomere-binding and terminal proteins and, therefore, most probably functions in replication of the linear plasmids (Bao and Cohen 2003; see also Chen, in this volume). R4, the most elaborate region of homology between pBD2 and pREL1, and region R5 are not conserved on pFiD188 nor on pRHL2. The gene products encoded by these two regions are involved in lipoprotein processing, and heavy metal and arsenate resistance (Sekine et al. 2006). In R.fascians strain D188, heavy metal resistance (cadmium) is carried on a separate replicon, the circular plasmid pD188 (Desomer et al. 1988). Interestingly, whereas one end of the four linear replicons is very conserved, the other end is unique for each plasmid (Fig. 2) and represents genes involved in the characteristic features of the particular strains. For plasmids pBD2 and pREL1, the unique end encodes specific metabolic functions, isopropylbenzene catabolism (Stecker et al. 2003) and alkane degradation (Sekine et al. 2006), respectively. For pFiD188, this region is possibly involved in virulence, whereas for pRHL2 it mainly contains hypothetical proteins. Besides the unique end, each of the four replicons has two additional nonconserved regions (Fig. 2). In pFiD188, the unique region U1 consists of four loci, three of which (hyp, att, and fas) have been identified as virulence loci through random insertion mutagenesis (Crespi et al. 1992); region U2 carries the nrp locus, and the terminal region

U3 harbors the stk locus. Although the latter unique regions have not been extensively characterized, they plausibly also encode functions that are important during the interaction of R.fascians with its host. In the next section, the genes located in the three unique regions of pFiD188 will be discussed in detail.

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