Several linear megaplasmids were detected in the facultatively lithoautotrophic Gram-positive bacterium Nocardia opaca The wild-type strain M R l l contains, in addition to the cccDNA plasmids pHG31-a and pHG31-b, the linear plasmids pHG201(270 kb), pHG202 (400 kb) and pHG203 (420 kb). The wild-type strain MR22 contains, in addition to the cccDNA plasmid pHG33, the linear plasmids pHG204 (180 kb), pHG205 (280 kb) and pHG206 (510 kb). After preparation of DNA from cells embedded in agarose, the linear plasmids were demonstrated by pulsed-field electrophoresis. By means of DNA probes for genes of soluble hydrogenase and ribulose-bisphosphate carboxylase, the conjugative plasmids pHG201 and pHG205 were shown to be the carriers of the genetic information for these enzymes. A restriction map of pHG201 for the enzymes AsnI, SpeI, XbaI is presented.
Rhodococcus fascians is currently the only phytopathogen of which the virulence genes occur on a linear plasmid. To get insight into the origin of this replicon and into the virulence strategy of this broad-spectrum phytopathogen, the sequence of the linear plasmid of strain D188, pFiD188, was determined. Analysis of the 198,917 bp revealed four syntenic regions with linear plasmids of R. erythropolis, R. jostii, and R. opacus, suggesting a common origin of these replicons. Mutational analysis of pFi_086 and pFi_102, similar to cutinases and type IV peptidases, respectively, showed that conserved region R2 was involved in plasmid dispersal and pointed toward a novel function for actinobacterial cutinases in conjugation. Additionally, pFiD188 had three regions that were unique for R. fascians. Functional analysis of the stk and nrp loci of regions U2 and U3, respectively, indicated that their role in symptom development was limited compared with that of the previously identified fas, att, and hyp virulence loci situated in region U1. Thus, pFiD188 is a typical rhodococcal linear plasmid with a composite structure that encodes core functions involved in plasmid maintenance and accessory functions, some possibly acquired through horizontal gene transfer, implicated in virulence and the interaction with the host.
As described previously, in Rhodococcus sp. (formerly Nocardia opaca) strains MRll and MR22, the ability to grow as an aerobic hydrogen bacterium (the Aut character) is located on giant conjugative linear plasmids -on pHG201 (270 kb) in strain MRll and on pHG205 (280 kb) in strain MR22. In an autotrophic transconjugant originating from MR22 a smaller plasmid, pHG207 (225 kb), was detected and shown to be a recombination product of the wild-type plasmids pHG204 and pHG205. A donor carrying pHG207 as the sole plasmid transferred the Aut marker at a 1000-fold frequency compared to the wild-type plasmid pHG205. Analysis of the plasmid ends revealed that plasmid pHG207 carries proteins at both ends; the proteins are linked to the 5' ends of the strands. The cloned end fragments of about 2 kb were sequenced and found to contain highly homologous sequences within the terminal 583 bp (left end part) and 560 bp (right end part). Several potential reading frames were detected, but database searching gave no indication about possibIe functions.
The telomers of several linear plasmids o f Rhodococcus opacus (formerly Nocardia opaca) were studied. The plasmids pHG201, pHG204 and pHG205 carry proteins bound to their ends, as shown by gel retardation experiments. A sequence hybridizing with the terminal sequence of pHG207, a recombinant linear plasmid consisting of the left part o f pHG204 and the right part of pHG205, which was analysed in a previous study by the authors, could be detected in all linear plasmids of the wild-type R. opacus strains MR11 and MR22. However, only pHG204 and pHG206 carry terminal inverted repeats (TIRs) like pHG207. Cloning and sequencing of the terminal fragment of pHG204 revealed a nearly perfect TIR of 1016 bp. In contrast, the termini of pHG2Ol and pHG205 share little homology. Sequence analysis of the two end fragments of pHG201 revealed a similarity of only 65% within the terminal 34/32 bp and a perfect TIR of only 3 bp. The results support the assumption that long TlRs are not absolutely necessary for replication and maintenance of linear plasmids.
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