Isolation and Characterization of a Rolling-Circle-Type Plasmid from Rhodococcus erythropolis and Application of the Plasmid to Multiple-Recombinant-Protein Expression

Abstract

We isolated, sequenced, and characterized the cryptic plasmid pRE8424 from Rhodococcus erythropolis DSM8424. Plasmid pRE8424 is a 5,987-bp circular plasmid; it carries six open reading frames and also contains cis-acting elements, specifically a single-stranded origin and a double-stranded origin, which are characteristic of rolling-circle-replication plasmids. Experiments with pRE8424 derivatives carrying a mutated single-stranded origin sequence showed that single-stranded DNA intermediates accumulated in the cells because of inefficient conversion from single-stranded DNA to double-stranded DNA. This result indicates that pRE8424 belongs to the pIJ101/pJV1 family of rolling-circle-replication plasmids. Expression vectors that are functional in several Rhodococcus species were constructed by use of the replication origin from pRE8424. We previously reported a cryptic plasmid, pRE2895, from R. erythropolis, which may replicate by a -type mechanism, like ColE2 plasmids. The new expression vectors originating from pRE8424 were compatible with those derived from pRE2895. Coexpression experiments with these compatible expression vectors indicated that the plasmids are suitable for the simultaneous expression of multiple recombinant proteins.Rhodococcus erythropolis is a gram-positive, high-GϩC-content bacterium (Actinobacteria) that can grow at temperatures ranging from 4 to 35°C (39). Because many Rhodococcus strains have broad metabolic diversity and an array of unique enzymatic capabilities, they are of interest for pharmaceutical, environmental, chemical, and energy studies as well as for applications in the desulfurization of fossil fuels (25) and the industrial production of acrylamide (21). Several researchers have developed various genetic tools to analyze Rhodococcus (9), including Escherichia coli-Rhodococcus shuttle vectors (5) and a gene disruption system (38).We have developed inducible expression vectors (pTip vectors) that work in several Rhodococcus species (26). The pTip vectors are tightly regulated expression vectors containing a tipA promoter (P tipA ) (4, 14), from which protein expression is induced by the antibiotic reagent thiostrepton (26). In our previous report, we showed that pTip vectors mediate heterologous and homologous protein expression, as they contain multiple cloning sites for 11 restriction enzyme sites and a hexahistidine (six-His) tag sequence and they work over a wide temperature range, from 4 to 35°C. The expression yields of recombinant proteins can be up to 10 mg per liter of R. erythropolis culture. In addition, some proteins that could not be expressed in E. coli were successfully expressed in R. erythropolis.The pTip vectors carry the repAB operon of the cryptic plasmid pRE2895, which is necessary for the autonomous replication of the plasmids in Rhodococcus cells. The repAB operon encodes the replication proteins RepA and RepB, which are characteristic of pAL5000-type plasmids (26,35). The replication mechanisms of pAL5000 and pRE2895 are unknown, but RepA proteins of...

A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

“…The hsaA gene was amplified using a forward primer (GCAATAGCATATG-CATCACCATCACCATCACATCGAAGGTAGGACGTCC-ATTCAACACGTGATG), which introduced an NdeI site, six codons for histidine, and a Factor Xa recognition site, and a reverse primer (GCCAAGCTTGGTAGCTGTCATTTCG-CCTAGACC), which introduced a HindIII site. The amplicon was first cloned into pET41bϩ (Novogen, Oakville, Ontario) as an NdeI/HindIII fragment, its nucleotide sequence was verified, and then subcloned in pTipQC2 (33) using the same restriction sites, yielding pTipHA. The hsaB gene was amplified using oligonucleotides (CTAGCTAGCGC-TCAGATCGATCCACGCACG and GCCAAGCTTCGTCT-GCTCGCGCCACTAG) that introduced NheI and HindIII sites flanking the gene.…”

Advances in the Development of Genetic Tools for the Genus Rhodococcus

“…The pTip/Nit vectors can replicate not only in R. erythropolis but also in R. fascians and R. opacus (transformation efficiencies are 3.8×10 5 , 8.2×10 2 , and 1.6×10 4 , respectively), but not in R. rhodochrous or R. ruber 24) . In contrast, it has been reported that several plasmids are maintained in broad host ranges, and these have been used for the construction of Rhodococcus-Escherichia coli shuttle vectors.…”

“…Several types of expression vectors for Rhodococcus have been developed by using two different replication origins 23,24) . One type of these vectors propagates via a q-type mechanism, while another type propagates via a rolling circle mechanism.…”

“…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.…”

Monooxygenases involved in the n-alkanes metabolism by Rhodococcus sp. BCP1: molecular characterization and expression of alkB gene

“…During the last decade, the following efforts have been directed towards the development of genetics strategies for the manipulation of Rhodococcus members: the construction of E.coliRhodococcus shuttle vectors [272][273][274], the development of transposome systems to create random transposon libraries [271,275,276] and unmarked mutagenesis deletion systems with SacB as counter selection to generate mutants as result of two consecutive DNA single crossover events [277][278][279]. Additionally, the following approaches have been recently applied in…”