Summary auto-generated
This study characterized the 5.9 kb cryptic plasmid pFAJ2600 isolated from Rhodococcus erythropolis NI86/21 by determining its complete nucleotide sequence. The plasmid contains eight open reading frames, including replication genes repA and repB that are related to the pAL5000-family of small replicons found in various actinomycetes. Additional genes identified include parA (encoding a partitioning ATPase), pmrA (a putative site-specific recombinase for multimer resolution), and several genes of unknown function. The origin of replication resembles that of ColE2 plasmids from enterobacteria, and the replication termination region shows similarities to Bacillus subtilis terminators. Using pFAJ2600 as a backbone, the researchers constructed E. coli-Rhodococcus shuttle vectors by inserting a chloramphenicol resistance marker. The resulting vectors, particularly pFAJ2574, were stably maintained without antibiotic selection in multiple Rhodococcus species including R. erythropolis, R. fascians, R. rhodochrous, and R. ruber, demonstrating broader host range than previously available shuttle vectors.
Key findings
- pFAJ2600 belongs to the pAL5000-related family of small replicons with repA and repB genes showing homology to both actinomycete plasmids and enterobacterial ColE2-type plasmids
- The plasmid encodes dual stabilization mechanisms: PmrA (a λ integrase-family site-specific recombinase for multimer resolution) and ParA (an ATPase for active partitioning)
- Shuttle vector pFAJ2574 was stably maintained in R. erythropolis, R. fascians, R. rhodochrous, and R. ruber without selective pressure, providing broader host range than previous Rhodococcus vectors
- The putative origin of replication shares structural features with ColE2 plasmids, and a potential termination region shows similarity to Bacillus subtilis replication terminators
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Abstract
The complete nucleotide sequence of the 5936 bp cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 was determined. Based on the characteristics of its putative replication genes, repA and repB, pFAJ2600 was assigned to the family of pAL5000-related small replicons identified in Mycobacterium (pAL5000), Corynebacterium (pXZ10142), Brevibacterium (pRBL1), Bifidobacterium (pMB1) and Neisseria (pJD1). The replication systems of these plasmids show striking similarities to the ones used by the ColE2 family of plasmids from Enterobacteria with respect to both trans-acting factors and ori sequences. Two possible plasmid stabilization systems are encoded on pFAJ2600: a site-specific recombinase (PmrA) related to the Escherichia coli Xer proteins for plasmid multimer resolution and an ATPase (ParA) related to the A-type of proteins in sop/par partitioning systems. The proposed replication termination region of pFAJ2600 has features in common with the Ter loci of Bacillus subtilis. Chimeras composed of a pUC18-Cmr derivative inserted in the parA-repA intergenic region of vector pFAJ2600 produced vectors that could be shuttled between Escherichia coli and several Rhodococcus species (R. erythropolis, R. fascians, R. rhodochrous, R. ruber). The pFAJ2600-based shuttle vector pFAJ2574 was stably maintained in R. erythropolis and R. fascians growing under non- selective conditions.