The terminal structures of linear plasmids from Rhodococcus opacus

Abstract

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.

Genomics ofActinobacteria: Tracing the Evolutionary History of an Ancient Phylum

“…Using pulsed-field gel electrophoresis, the genomes of some other, still-unsequenced mycelial Actinobacteria taxa, such as Actinomyces, Amycolatopsis, Actinoplanes, Streptoverticillium, and Micromonospora, were also shown to be linear, with sizes ranging from 7.7 Mb (e.g., Micromonospora chalcea) to 9.7 Mb (Streptoverticillium abikoense), while sometimes also harboring large linear plasmids (362). Linear plasmids, typically possessing short inverted repeats at their termini and protein-bound 5Ј ends, are often present in Actinobacteria (216).…”

Functional Characterization of a Catabolic Plasmid from Polychlorinated- Biphenyl-Degrading Rhodococcus sp. Strain RHA1

“…2A, to telomeric regions of pHG207 of Rhodococcus sp. strain MR2253 (30) and to the right telomeres of pRHL2 (56), pBD2 of R. erythropolis (60), pHG201 of R. opacus MR11, and pHG204 of R. opacus MR22 (31). Each of these telomere sequences contains two sets of inverted repeats flanking the GCTXCGC central motif ( Fig.…”

A linear megaplasmid, p1CP, carrying the genes for chlorocatechol catabolism of Rhodococcus opacus 1CP

“…However, manual alignment of the terminal 100-117 nucleotides of all hitherto available ends of linear Rhodococcus plasmids clearly confirmed the presence of strongly conserved palindromic motifs. Two copies of the motif GCTXCGC were found in each terminus and it was already assumed earlier that these structures are essential for replication (Kalkus et al, 1998;Shimizu et al, 2001). Essentially the same central motif with the potential to form a stable single C-residue loop close to the sheared T : C pairing was found in the linear Streptomyces plasmids pSCL1 (Wu & Roy, 1993), pSLA2 (Hirochika et al, 1984) and SLP2 , as well as in the terminal segments of several linear Streptomyces chromosomes (Huang et al, 1998).…”

“…However, in p1CP-J the palindrome of position 12-34 is imperfect because of a mismatch at position 14. Both palindromic structures contain the central motif GCTXCGC, already obtained for other plasmid termini of rhodococci (Kalkus et al, 1998;Shimizu et al, 2001) and streptomycetes (Bey et al, 2000;Huang et al, 1998). Several other regions of striking similarity were identified (alignment positions 1-2, 5-7 and 49-62).…”

“…One terminal fragment was identified by hybridization of SstII-digested purified p1CP DNA using a DIG-labelled 2?3 kb SalI fragment of the right end of the linear Aut plasmid pHG201 from R. opacus MR11 (Kalkus et al, 1998). Ligation into SstII/EcoRV-digested pBluescript II KS(+) and transformation into E. coli DH5a, followed by colony hybridization, led to the identification of one positive clone, p1CP-S, containing a 1?56 kb SstII/blunt-end insert.…”