A small cryptic plasmid from Rhodococcus erythropolis: characterization and utility for gene expression

Abstract

Exploration of metabolically diverse rhodococci is generally hampered by the lack of genetic tools. A small cryptic plasmid (pAN12) isolated from Rhodococcus erythropolis strain AN12 was sequenced. Plasmid pAN12 encodes proteins that share homology to replication proteins and putative cell division proteins. Based on in vitro transposon mutagenesis, we determined that the Rep protein of pAN12 is essential for plasmid replication in Rhodococcus spp., and the putative cell division protein Div is important for plasmid stability. The pAN12 replicon is able to replicate in R. erythropolis strains AN12 and CW23 (ATCC 47072) and is compatible with the nocardiophage Q4 replicon present on a Rhodococcus shuttle plasmid pDA71. pAN12 appears to belong to the pIJ101/pJV1 family of rolling circle replication plasmids. Expression of an isoprenoid pathway gene ( dxs) on the pAN12-derived multicopy shuttle vector increased production of carotenoid pigments in R. erythropolis ATCC 47072.

TraA is required for megaplasmid conjugation in Rhodococcus erythropolis AN12

“…AN12 electrocompetent cells were prepared essentially as previously described (Kostichka et al, 2003), except cells were grown in NBYE/0.05% Tween 80 media in a 1 L baZed Xask with shaking until OD 600 of about 0.5 was reached. Standard transformations with plasmids capable of replicating in Rhodococcus was achieved by incubating 0.5 g of transforming DNA in 1£ TE buVer with 100 to 150 l competent AN12 cells for 5 min prior to electroporation.…”

“…Rhodococcus genomic DNA was prepared as previously described . Rhodococcus plasmid DNA was isolated using the procedures previously described (Kostichka et al, 2003). pEZTn1F6 and pEZTn5A6 were generated via a plasmid rescue approach by digesting 2 g total genomic DNA prepared from AN12PL-1F6 and AN12-5A6, respectively, with EcoRI for 2 h, followed by incubation at 65°C for 30 min to abolish EcoRI activity.…”

“…6.3 kb, called pAN12 (Kostichka et al, 2003). Preliminary experiments in our lab suggested that at least two diVerent species of megaplasmids also exist in the AN12 genome.…”

“…Besides the initial characterization of its small cryptic plasmid, pAN12 (Kostichka et al, 2003), it was not known whether the genome of AN12 harbored other extrachromosomal replicons, nor whether any of its replicons are transmissible via bacterial conjugation. We show in this study that AN12 possesses at least three distinctly migrating species of megaplasmids, and that at least two of the AN12 megaplasmids (pREA400 and pREA250) can be mobilized to a closely related R. erythropolis strain, SQ1.…”

Advances in the Development of Genetic Tools for the Genus Rhodococcus

“…Data accumulated from basic studies on the characteristic features of rhodococci and the increasing knowledge obtained from the genome databases have enhanced the development of genetic manipulation techniques used for rhodococci analysis. Such techniques include the development of shuttle vectors using cryptic and/or antibiotic-resistant plasmids derived from Rhodococcus strains 10,19,20) , efficient transformation techniques using electroporation 21,22) , the expression vectors for protein production 23,24) , random mutagenesis methods using transposons or spontaneous illegitimate recombination 25,26) , and targeted gene disruption systems 11,27) . These techniques along with increasing genome information are enabling cell engineering in a manner such that there is an improvement in the useful properties of rhodococci such as their ability to act as biocatalysts; this improvement is due to the activation of catabolic pathways and/or depletion of the undesirable genes.…”