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At least 19 recordsLinked to original sources
PubMed1988

Construction of an Escherichia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp.

A plasmid transformation system for Rhodococcus sp. strain H13-A was developed by using an Escherichia coli-Rhodococcus shuttle plasmid constructed in this study. Rhodococcus sp. strain H13-A contains three cryptic indigenous plasmids, designated pMVS100, pMVS200, and pMVS300, of 75, 19.5, and 13.4 kilobases (kb), respectively. A 3.8-kb restriction fragment of pMVS300 was cloned into pIJ30, a 6.3-kb pBR322 derivative, containing the E. coli origin of replication (ori) and ampicillin resistance determinant (bla), as well as a Streptomyces gene for thiostrepton resistance, tsr. The resulting 10.1-kb recombinant plasmid, designated pMVS301, was isolated from E. coli DH1(pMVS301) and transformed into Rhodococcus sp. strain AS-50, a derivative of strain H13-A, by polyethylene glycol-assisted transformation of Rhodococcus protoplasts and selection for thiostrepton-resistant transformants. Thiostrepton-resistant transformants were also ampicillin resistant and were shown to contain pMVS301, which was subsequently isolated and transformed back into E. coli. The cloned 3.8-kb fragment of Rhodococcus DNA in pMVS301 contains a Rhodococcus origin of replication, since the hybrid plasmid was capable of replication in both genera. The plasmid was identical in E. coli and Rhodococcus transformants as determined by restriction analysis and was maintained as a stable, independent replicon in both organisms. Optimization of the transformation procedure resulted in transformation frequencies in the range of 10(5) transformants per micrograms of pMVS301 DNA in Rhodococcus sp. strain H13-A and derivative strains. The plasmid host range extends to strains of Rhodococcus erythropolis, R. globulerus, and R. equi, whereas stable transformants were not obtained with R. rhodochrous or with several coryneform bacteria tested as recipients. A restriction map demonstrated 14 unique restriction sites in pMVS301, some of which are potentially useful for molecular cloning in Rhodococcus spp. and other actinomycetes. This is the first report of plasmid transformation and of heterologous gene expression in a Rhodococcus sp.

Ampicillin↗
PubMed1995

An improved Escherichia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp. using electroporation.

The genetic studies of metabolically diverse Rhodococcus spp. have been hampered by the lack of a system of introducing exogenous DNA. The authors improved an existing Escherichia coli-Rhodococcus shuttle vector (pMVS301) by removing much of the DNA not needed for replication and adding a multicloning site. This improved vector (pBS305) is 7.9 kb in length. Its ability to transform Rhodococcus was tested using electroporation parameters optimized for introduction of pMVS301 into Rhodococcus. Transformation efficiencies as high as 10(5) cfu micrograms-1 DNA were obtained although efficiencies varied depending on the Rhodococcus strain tested. The improved vector pBS305 offers great utility for genetic studies of Rhodococcus because its small size enables movement of large inserts of DNA into Rhodococcus, it has multicloning sites, contains a highly selective thiostrepton marker, and can be replicated in both E. coli and Rhodococcus.

DNA, Bacterial↗
PubMed2002

Transfer of Tsukamurella wratislaviensis Goodfellow et a. 1995 to the genus Rhodococcus as Rhodococcus wratislaviensis comb. nov..

A polyphasic study was undertaken to clarify the taxonomic position of the type strain (N805T) of Tsukamurella wratislaviensis. This organism showed a combination of phenotypic properties, notably chemotaxonomic markers, consistent with its classification in the genus Rhodococcus. Comparative 16S rDNA sequencing studies indicated that strain 805T falls into the Rhodococcus erythropolis subclade, where it forms a monophyletic group with the type strains of Rhodococcus opacus and Rhodococcus percolatus. The close relationship between these strains was underpinned by the results of mycolic acid analyses. However, strain N805T was distinguished from the R. opacus and R. percolatus strains in DNA-DNA pairing experiments and by using a range of phenotypic properties. In light of these studies, it is clear that strain N805T is misclassified in the genus Tsukamurella. It is, therefore, proposed that Tsukamurella wratislaviensis Goodfellow et al. 1995 be transferred to the genus Rhodococcus as Rhodococcus wratislaviensis comb. nov..

Actinomycetales↗
PubMed1995

Cloning and expression of the s-triazine hydrolase gene (trzA) from Rhodococcus corallinus and development of Rhodococcus recombinant strains capable of dealkylating and dechlorinating the herbicide atrazine.

We used degenerate oligodeoxyribonucleotides derived from the N-terminal sequence of the s-triazine hydrolase from Rhodococcus corallinus NRRL B-15444R in an amplification reaction to isolate a DNA segment containing a 57-bp fragment from the trzA gene. By using the nucleotide sequence of this fragment, a nondegenerate oligodeoxyribonucleotide was synthesized and used to screen a genomic library of R. corallinus DNA for fragments containing trzA. A 5.3-kb PstI fragment containing trzA was cloned, and the nucleotide sequence of a 2,450-bp region containing trzA was determined. No trzA expression was detected in Escherichia coli or several other gram-negative bacteria. The trzA gene was subcloned into a Rhodococcus-E. coli shuttle vector, pBS305, and transformed into several Rhodococcus strains. Expression of trzA was demonstrated in all Rhodococcus transformants. Rhodococcus sp. strain TE1, which possesses the catabolic gene (atrA) for the N-dealkylation of the herbicides atrazine and simazine, was able to dechlorinate the dealkylated metabolites of atrazine and simazine when carrying the trzA gene on a plasmid. A plasmid carrying both atrA and trzA was constructed and transformed into three atrA- and trzA-deficient Rhodococcus strains. Both genes were expressed in the transformants. The s-triazine hydrolase activity of the recombinant strains carrying the trzA plasmid were compared with that of the R. corallinus strain from which it was derived.

Amino Acid Sequence↗
PubMed1994

Cesium Accumulation and Growth Characteristics of Rhodococcus erythropolis CS98 and Rhodococcus sp. Strain CS402.

Growth and cesium accumulation characteristics of two cesium-accumulating bacteria isolated from soils were investigated. Rhodococcus erythropolis CS98 and Rhodococcus sp. strain CS402 accumulated high levels of cesium (approximately 690 and 380 mumol/g [dry weight] of cells or 92 and 52 mg/g [dry weight] of cells, respectively) after 24 h of incubation in the presence of 0.5 mM cesium. The optimum pH for cesium uptake by both Rhodococcus strains was 8.5. Rubidium and cesium assumed part of the role of potassium in the growth of both Rhodococcus strains. Potassium and rubidium inhibited cesium accumulation by these Rhodococcus strains. It is likely that both Rhodococcus strains accumulated cesium through a potassium transport system.

Journal Article↗
PubMed1998

Cloning of new Rhodococcus extradiol dioxygenase genes and study of their distribution in different Rhodococcus strains.

Four extradiol dioxygenase genes which encode enzymes active against catechol and substituted catechols were cloned from two different Rhodococcus strains, and their nucleotide sequences were determined. A catechol 2,3-dioxygenase gene (edoC) was shown to be identical to the previously described ipbC gene from the isopropylbenzene operon of Rhodococcus erythropolis. Amino acid sequences deduced from the three other genes (edoA, edoB and edoD) were shown to have various degrees of homology to different extradiol dioxygenases. The EdoA and EdoB dioxygenases were classified as belonging to the third family of type I oxygenases and represented two new subfamilies, whereas the EdoD dioxygenase was a type II enzyme. Analysis of six Rhodococcus strains revealed a wide distribution of the above dioxygenase genes. Rhodococcus sp. 11 was shown to harbour all four of the analysed dioxygenase genes. Nucleotide sequences homologous to the edoB gene were present in all of the strains, including R. erythropolis NCIMB 13065, which did not utilize any of the aromatic compounds analysed. The latter finding points to the existence of a silent pathway(s) for degradation of aromatic compounds in this Rhodococcus strain.

Amino Acid Sequence↗
PubMed2026

Rhodococcus dendrobeaniae sp. nov., an actinomycete isolated from an Arctic marine invertebrate exhibiting cytotoxic activity, and an emended description of Rhodococcus sovatensis.

A polyphasic study was conducted to establish the taxonomic status of strain T060T, an orange, aerobic, coccoid and non-motile actinomycete, isolated from a marine bryozoan (Dendrobeania sp.) collected in the Barents Sea. Phylogenetic analysis of the 16S rRNA gene sequences revealed Rhodococcus sovatensis DSM 102881T as the closest related species to strain T060T with a similarity of 99.54%. Phylogenomic analysis confirmed a close relationship between T060T and R. sovatensis DSM 102881T, while supporting their distinction. Digital DNA-DNA hybridization and average nucleotide identity values between strain T060T and R. sovatensis DSM 102881T were 26.4 and 84.3%, respectively, supporting the delineation of the isolate as a new species. Genomic characterization of the assembled genomes of T060T and R. sovatensis DSM 102881T showed genome sizes of 5.3 and 4.2 Mbp, with a G + C content of 64.38 and 65.01%, respectively. Genome analysis of strain T060T identified 15 biosynthetic gene clusters (BGCs) with low sequence similarity to known BGCs, indicating its capacity to produce unknown, potentially bioactive secondary metabolites. Furthermore, growth of T060T in eight different media revealed condition-dependent cytotoxic activity. The strongest cytotoxicity was observed for fractionated extracts from T060T grown in half-strength ISP 2 with filtered seawater against the human malignant cell lines MCF7 and A2058, and to a lesser extent against the non-malignant MRC5 cell line, highlighting its biotechnological potential. Based on the data from polyphasic taxonomy studies, it is proposed that strain T060T be classified in the genus Rhodococcus as Rhodococcus dendrobeaniae sp. nov. Additionally, the acquired data of R. sovatensis were used to amend its original description.

Rhodococcus↗
PubMed2004

Degradation of estrogens by Rhodococcus zopfii and Rhodococcus equi isolates from activated sludge in wastewater treatment plants.

We have isolated four strains of Rhodococcus which specifically degrade estrogens by using enrichment culture of activated sludge from wastewater treatment plants. Strain Y 50158, identified as Rhodococcus zopfii, completely and rapidly degraded 100 mg of 17beta-estradiol, estrone, estriol, and ethinyl estradiol/liter, as demonstrated by thin-layer chromatography and gas chromatography-mass spectrometry analyses. Strains Y 50155, Y 50156, and Y 50157, identified as Rhodococcus equi, showed degradation activities comparable with that of Y 50158. Using the random amplified polymorphism DNA fingerprinting test, these three strains were confirmed to have been derived from different sources. R. zopfii Y 50158, which showed the highest activity among these four strains, revealed that the strain selectively degraded 17beta-estradiol during jar fermentation, even when glucose was used as a readily utilizable carbon source in the culture medium. Measurement of estrogenic activities with human breast cancer-derived MVLN cells showed that these four strains each degraded 100 mg of 17beta-estradiol/liter to 1/100 of the specific activity level after 24 h. It is thus suggested that these strains degrade 17beta-estradiol into substances without estrogenic activity.

Biodegradation, Environmental↗
PubMed1992

Overexpression of a Rhodococcus erythropolis protein in Escherichia coli with immunological identity to the Rhodococcus steroid 1-dehydrogenase. Immunoelectron microscopic localization and electrophoretic studies.

The recombinant Escherichia coli K-12 strain chi 6060 harbouring the plasmid pYA 1201 with a gene from Rhodococcus erythropolis IMET 7030 overexpressed a protein which reacts with a monospecific antiserum against the steroid 1-dehydrogenase (Sdh) from the same Rhodococcus strain. It was shown previously that this recombinant protein exhibits no enzymatic activity. By immunogold labelling the protein was localized on ultrathin sections of the recombinant E. coli strain. After cultivation at 37 degrees C it was found within large cytoplasmic compartments (inclusion bodies). The inclusion bodies occupied 40% to 75% of the sectioned cell area. The highest amount of protein was observed after induction of the culture with isopropyl-beta-D-thiogalactopyranoside. Approximately 20% of the induced cells became enlarged (up to 5-fold of the normal size) and deformed; multiplication of the Rhodococcus protein producing cells was inhibited. After ultrasonic cell disintegration the inclusion bodies were found only in the fraction of the sedimented cell debries and did still react with anti-Sdh. When recombinant E. coli cells were cultivated at 28 degrees C, inclusion bodies appeared very seldom and the immunoreactive protein was distributed throughout the whole cytoplasm.

Animals↗
PubMed2006

Analysis of the 7.6-kb cryptic plasmid pNC500 from Rhodococcus rhodochrous B-276 and construction of Rhodococcus-E. coli shuttle vector.

Four circular cryptic plasmids were detected from propene-degrading Rhodococcus rhodochrous (formerly Nocardia corallina) B-276 and the smallest 7.6-kb plasmid, named pNC500 was used to construct Rhodococcus-E. coli shuttle vector, pNC5403. Sequence analysis of pNC500 revealed that the plasmid contains eight potential ORFs, namely 1 through 8. The deduced amino acid sequences for ORFs 3, 4, 6, and 7 show homology with those of Rep A, Rep B, DNA methyl-transferase (M.XamI), and restriction nuclease (R.XamI), respectively. The region responsible for replication in the potent oil-desulfurization bacterium, Rhodococcus opacus T09 was determined as 3.7 kb-XbaI/BalI fragment which contains ORFs 3 and 4, while no transformants were obtained when ORF 4 was partially deleted, suggesting that both are required for its replication. Alignment of the predicted amino acid sequences revealed that ORFs 3 and 4 were DNA binding protein and DNA primase, respectively. A compatibility test with pAL5000-related plasmid vector, pRHK1, which contains pRC4, revealed that pNC5403 was compatible with pRHK1 suggesting that each replication origin would be different. ORFs 3 and 4 containing a pNC5403 derivative, pN5DXB, was stably maintained for over 80 generations in the absence of antibiotic selective conditions.

Amino Acid Sequence↗
PubMed1998

Overexpression of high-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1 in recombinant Rhodococcus cells.

High-molecular-mass nitrile hydratase (H-NHase, 530 kDa) is a cobalt-containing enzyme produced by Rhodococcus rhodochrous J1. For efficient production of H-NHase in R. rhodochrous ATCC12674, several plasmids were constructed. The enzyme was produced in the recombinant Rhodococcus cells only in the presence of an upstream region (approximately 4 kb) of the H-NHase gene under the control of the promoter for the amidase-NHase gene cluster from Rhodococcus sp. N-774. Although H-NHase was produced as a soluble protein in the cells, the protein did not show NHase activity. However, when the recombinant R. rhodochrous ATCC12674 cells were cultured in the presence of amide compounds, such as crotonamide and methacrylamide, markedly high NHase activity was detected, Gel-filtration chromatography revealed that the NHases produced by the cells grown in the presence and absence of the amide compounds had a molecular mass of more than 500 kDa and 50-80 kDa respectively. These results suggest that the amide compounds are essential for subunit assembly to form an enzymatically active multimer. By the use of the recombinant expression system, NHase activity 1.7 times higher than that of the original strain, R. rhodochrous J1, was achieved.

Dose-Response Relationship, Drug↗
PubMed1995

Brevibacterium linens pBL33 and Rhodococcus rhodochrous pRC1 cryptic plasmids replicate in Rhodococcus sp. R312 (formerly Brevibacterium sp. R312).

The replication of two cryptic plasmids from Brevibacterium linens ATCC 9174 (pBL33) and Rhodococcus rhodochrous ATCC 4276 (pRC1) was investigated in Rhodococcus sp. R312 (formerly Brevibacterium sp. R312). The recombinant plasmids pSP33 (pBL33 derivative) and pSPC1 (pRC1 derivative) were found to be suitable for establishing new host-vector systems for Rhodococcus sp. R312. They all carry the Tn903 neomycin-resistance-encoding gene (aphI).

Bacterial Proteins↗
PubMed2001

Preparative isolation of lipid inclusions from Rhodococcus opacus and Rhodococcus ruber and identification of granule-associated proteins.

Triacylglycerol granules synthesized and accumulated by Rhodococcus opacus and Rhodococcus ruber were isolated by glycerol density gradient centrifugation. Whereas only one type of granule could be isolated from R. opacus, two types of granules with different specific densities were isolated from R. ruber. Both types of R. ruber granules showed a similar content of triacylglycerols and poly(3-hydroxybutyrate- co-3-hydroxyvalerate), but the protein profiles of both types were significantly different. The granules with the lower specific density were colorless; the granules with the higher specific density had a deep orange pigmentation. Solubilization studies revealed three different groups of granule-associated proteins: (1) unspecifically bound proteins, (2) relatively weakly associated proteins, and (3) proteins that resisted solubilization by treatment with 2 M NaCl, 2% (w/v) Triton X-114, 6 M guanidinium hydrochloride, up to 8% (w/v) SDS, and proteolytic digestion. The strong association of proteins of the last group suggested that these may play a specific role in the synthesis or mobilization of storage lipids or in the structure of the granules. The N-terminal amino acid sequences of the most tightly bound proteins were obtained. Proteins of low molecular weight with striking sequence similarity to the ribosomal protein L7 from various actinomycetes were always copurified with the granules.

Amino Acid Sequence↗
PubMed2000

Accumulation and mobilization of storage lipids by Rhodococcus opacus PD630 and Rhodococcus ruber NCIMB 40126.

The time course of the accumulation of triacylglycerols (TAGs) in Rhodococcus opacus PD630 or of TAGs plus polyhydroxyalkanoates (PHA) in Rhodococcus ruber NCIMB 40126 with gluconate or glucose as carbon source, respectively, was studied. In addition, we examined the mobilization of these storage compounds in the absence of a carbon source. R. opacus accumulated TAGs only after the exhaustion of ammonium in the medium, and, with a fixed concentration of the carbon source, the amounts of TAGs in the cells increased with decreasing concentrations of ammonium in the medium. When these cells were incubated in the absence of an additional carbon source, about 90% of these TAGs were mobilized and used as endogenous carbon source, particularly if ammonium was available. R. ruber accumulated a copolyester consisting of 3-hydroxybutyrate and 3-hydroxyvalerate already during the early exponential growth phase, whereas TAGs were synthesized and accumulated mainly during the late exponential and stationary growth phases. In the stationary growth phase, synthesis of TAGs continued, whereas PHA was partially mobilized. In the absence of an additional carbon source but in the presence of ammonium, mobilization of TAGs started first and was then paralleled by the mobilization of PHA, resulting in an approximately 90% and 80% decrease of these storage compounds, respectively. During the accumulation phase, interesting shifts in the composition of the two storage compounds occurred, indicating that the substrates of the PHA synthase and the TAG synthesizing enzymes were provided to varying extents, depending on whether the cells were in the early or late exponential or in the stationary growth phase.

Culture Media↗
PubMed2004

Isolation and characterization of the Rhodococcus opacus thiostrepton-inducible genes tipAL and tipAS: application for recombinant protein expression in Rhodococcus.

We cloned the Rhodococcus opacus (strain DSM 44193) tipA gene, which encodes two translation products, TipAL and TipAS. The gene products are homologous to the Streptomyces spp. TipAL and TipAS proteins, respectively. The tipA promoter is highly active and TipAS protein is predominantly accumulated in R. opacus cells when the inducer of transcription, thiostrepton, was presented in culture medium. We found that thiostrepton is also induced the expression of an endogenous TipA-family protein in Rhodococcus erythropolis (strain JCM3201). The minimal tipA promoter region was defined (57 bp) and the conserved nucleotide sequence of the putative TipAL protein binding site (TipA-box) was identified in that region. The tipA gene is presumed to be transcribed into a leaderless mRNA. We applied the tipA promoter successfully for recombinant protein expression in R. erythropolis cells.

Artificial Gene Fusion↗
PubMed1997

Structural analysis of the 6 kb cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 and construction of Escherichia coli-Rhodococcus shuttle vectors.

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.

Amino Acid Sequence↗
PubMed1998

Characterization of the basic replicon of Rhodococcus plasmid pSOX and development of a Rhodococcus-Escherichia coli shuttle vector.

The replication region of a 100-kb desulfurization plasmid (pSOX) from Rhodococcus sp. strain X309 was localized to a 4-kb KpnI fragment, and its sequence was determined. The amino acid sequence of one of the predicted open reading frames (ORFs) was related to the putative replication (Rep) protein sequences of the mycobacterial pLR7 family of plasmids. Three of the five predicted ORF products were identified by radiolabelling with the Escherichia coli T7 polymerase/promoter system. In E. coli, the Rep protein of pSOX was apparently synthesized in a shortened form, 21.3 kDa instead of the predicted 41.3 kDa, as a result of an internal initiation. This situation is reminescent of that for some bacterial Rep proteins. A shuttle plasmid was constructed with the pSOX origin, pBluescript II KS-, and the chloramphenicol resistance (Cmr) gene from pRF29. This new shuttle plasmid was used to demonstrate expression of the Bacillus subtilis sacB gene in a strain of Rhodococcus, rendering it sensitive to the presence of sucrose.

Amino Acid Sequence↗
PubMed1999

Isolation and characterization of catechol 1,2-dioxygenases from Rhodococcus rhodnii strain 135 and Rhodococcus rhodochrous strain 89: comparison with analogous enzymes of the ordinary and modified ortho-cleavage pathways.

Catechol 1,2-dioxygenases of the ordinary ortho-cleavage pathway have been isolated from strains Rhodococcus rhodnii 135 and Rhodococcus rhodochrous 89 grown on phenol as the sole source of carbon and energy. The activities of the catechol 1,2-dioxygenases with 3- and 4-methylpyrocatechols were 1.3-1.5 times higher than those with pyrocatechol. The rate of oxidation of 3-chloropyrocatechol catalyzed by both enzymes was 20% of the rate of oxidation of unsubstituted pyrocatechol. The enzymes are homodimers composed of 37-kD subunits.

Catechol 1,2-Dioxygenase↗