Sequence analysis of three plasmids harboured in Rhodococcus erythropolis strain PR4

Rhodococcus erythropolis strain PR4 has been isolated as an alkane-degrading bacterium. The strain harbours one linear plasmid, pREL1 (271 577 bp) and two circular plasmids, pREC1 (104 014 bp) and pREC2 (3637 bp), all with some sequence similarities to other Rhodococcus plasmids. For pREL1, pREC1 and pREC2, 298, 102 and 3 open reading frames, respectively, were predicted. Linear plasmid pREL1 has several regions homologous to plasmid pBD2 found in R. erythropolis BD2. Sequence analysis of pREL1 and pBD2 identified common metal-resistance genes on both, but pREL1 also encodes alkane-degradation genes not found on pBD2, with enzyme constituents some of which are quite different from those of other organisms. The alkane hydroxylase consisted of a cytochrome P450 monooxygenase, a 2Fe-2S ferredoxin, and a ferredoxin reductase. The ferredoxin reductase amino acid sequence resembles the AlkT (rubredoxin reductase) sequence. A zinc-containing alcohol dehydrogenase further oxydizes alkanols, alkane oxidation products catalysed by alkane hydroxylase. Of the circular plasmids, the pREC1 sequence is partially similar to the sequence of pREAT701, the virulence plasmid found in Rhodococcus equi. pREC1 has no pREAT701 virulence genes and encodes genes for beta-oxidation of fatty acids. Thus, joint actions of enzymes encoded by pREL1 and pREC1 may enable efficient mineralization of alkanes.     

Molecular Genetic Markers for Identification of Rhodococcus erythropolis and Rhodococcus qingshengii

Microbiology - The application of restriction analysis of amplification products of the genes rpoC and alkB, encoding the synthesis of the DNA-dependent RNA polymerase β'-subunit and...     

An unusual formaldehyde oxidizing system in Rhodococcus erythropolis grown on compounds containing methyl groups

Abstract During utilization of compounds containing methyl groups, the non-methylotrophic bacteria Rhodococcus erythropolis oxidized the methyl groups entirely to carbon dioxide. This oxidation was linked to the presence of an NAD-dependent formaldehyde dehydrogenase activity which was lost on dialysis. The activity could be restored by the addition of boiled extract but not by adding the known cofactors glutathione or tetrahydrofolate.
A further dehydrogenase activity with formaldehyde as substrate was found in ethanolgrown cells. This activity could be differentiated from that in methyl group metabolizing cells.     

Establishment of an effective oligotrophic cultivation system for Rhodococcus erythropolis N9T-4

Rhodococcus erythropolis N9T-4 grows on an inorganic solid-state medium with no additional carbon and energy sources; however, it is unable to grow well in a liquid culture medium under the oligotrophic conditions. We examined submerged cultivations of N9T-4 using a polyurethane foam sponge to achieve approximately 10 times of the oligotrophic growth of the bacterium in the liquid culture medium.     

Enzymatic desulfurization of dibenzothiophene by a cell-free system of Rhodococcus erythropolis D-1

Abstract Pseudomonas syringae cells were exposed to Cu²⁺ alone or in the precence of acetate, proline or cysteine, at concentrations that reduced free Cu²⁺ to 1/10 of the total copper. Ligand concentrations (designated as isoeffective) were determined experimentally using a Cu²⁺-selective electrode and confirmed by computer calculations using published stability constants. Exposure of P. syringae cells to Cu²⁺ alone resulted in rapid and pronounced cell death, and binding of most of the copper in solution. The addition of acetate, proline or cysteine, a few minutes after Cu²⁺ treatment, resulted in a significant reduction in cell death, and in the amount of copper bound to the cells. For short exposures to Cu²⁺, cysteine was more effective than acetate or proline, but after 60 min of treatment, similar results were observed with these ligands. The addition of ligands before Cu²⁺ resulted in even more reduced copper toxicity. The results showed that, at isoeffective concentrations, weak and moderate copper-ligands can effectively antagonize copper toxicity, and that this protective effect does not require previously equilibrated copper-ligand solutions and is not very dependent of the nature of the ligand.     

Studies on utilization of acetonitrile by Rhodococcus erythropolis A10

A petrochemical wastewater isolate, capable of utilizing high concentrations of acetonitrile and acetamide as the sole source of carbon and nitrogen was identified as Rhodococcus erythropolis A10. Cell-free extracts of acetonitrile-grown cells exhibited activities corresponding to nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4), which mediate the two-step breakdown of acetonitrile into acetic acid and ammonia. Studies indicated that both these enzymes in R. erythropolis A10 are intracellular, inducible and capable of hydrolysing a wide range of nitriles, including simple (acetonitrile, propionitrile), branched-chain (isobutyronitrile) and dinitrile (succinonitrile). The specific activity of the amidase was found to be several-fold higher than nitrile hydratase.     

Biosensor of the reactor type based on Rhodococcus erythropolis HL TM-1 cells for determining 2,4-dinitrophenol

A model of a reactor-type biosensor based on the Rhodococcus erythropolis HL PM-1 was developed for amperometric detection of 2,4-dinitrophenol (2,4-DNP). The effects of the matrix material (agar and calcium alginate gels, ceramic support, and cellulose powder) on the biosensor signal concentration dependence, detection time, and biosensor stability were studied. In case of bacterial cells immobilized on cellulose powder, the lower limit of 2,4-DNP detection was 20 microM and the time of single analysis, the biosensor recovery included, was 30-50 min. In the continuous detection mode, the biosensor response was maintained at a stable level without biosensor inactivation for ten days. The biosensor can be used as an element of a complex analytical system for detecting nitroaromatic compounds in samples.     

TraA is required for megaplasmid conjugation in Rhodococcus erythropolis AN12

Pulsed-field gel electrophoresis (PFGE) revealed three previously uncharacterized megaplasmids in the genome of Rhodococcus erythropolis AN12. These megaplasmids, pREA400, pREA250, and pREA100, are approximately 400, 250, and 100kb, respectively, based on their migration in pulsed-field gels. Genetic screening of an AN12 transposon insertion library showed that two megaplasmids, pREA400, and pREA250, are conjugative. Mobilization frequencies of these AN12 megaplasmids to recipient R. erythropolis SQ1 were determined to be approximately 7x10(-4) and 5x10(-4) events per recipient cell, respectively. It is known for other bacterial systems that a relaxase encoded by the traA gene is required to initiate DNA transfer during plasmid conjugation. Sequences adjacent to the transposon insertion in megaplasmid pREA400 revealed a putative traA-like open reading frame. A targeted gene disruption method was developed to generate a traA mutation in AN12, which allowed us to address the role of the traA gene product for Rhodococcus megaplasmid conjugation. We found that the AN12 traA mutant is no longer capable of transferring the pREA400 megaplasmid to SQ1. Furthermore, we confirmed that the conjugation defect was specifically due to the disruption of the traA gene, as pREA400 megaplasmid conjugation defect is restored with a complementing copy of the traA gene.     

Production of 3,4-dihydroxyphthalate from phthalate by a membrane-bound two-enzyme system from Rhodococcus erythropolis

 Gram-positive Rhodococcus erythropolis strain S1 formed enzymes for the degradation of phthalate when grown in a phthalate-containing minimal medium. The membrane fraction prepared from phthalate-grown cells by ultrasonication converted phthalate to protocatechuate as the final product. Using two membrane-bound enzymes, phthalate 3,4-dioxygenase (PO) and 3,4-dihydro-3,4-dihydroxyphthalate 3,4-dehydrogenase (PH), prepared by solubilization of the membrane fraction, 3,4-dihydroxyphthalate was selectively obtained from phthalata. Fe²⁺ and Mn²⁺ stimulated the formation of 3,4-dihydroxyphthalate by the membrane-bound PO and PH system. Received: 27 April 1994/Received last revision: 19 August 1994/Accepted: 12 September 1994     

Rhodococcus erythropolis DCL14 contains a novel degradation pathway for limonene

Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample. This organism was identified as a strain of Rhodococcus erythropolis by chemotaxonomic and genetic studies. R. erythropolis DCL14 also assimilated the terpenes limonene-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (-)-menthol, while perillyl alcohol was not utilized as a carbon and energy source. Induction tests with cells grown on limonene revealed that the oxygen consumption rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of R. erythropolis DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1, 2-monooxygenase activity, a cofactor-independent limonene-1, 2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation studies showed that (1S,2S,4R)-limonene-1,2-diol, (1S, 4R)-1-hydroxy-2-oxolimonene, and (3R)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)-limonene degradation pathway. The opposite enantiomers [(1R,2R,4S)-limonene-1,2-diol, (1R, 4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxoheptanoate] were found in the (4S)-limonene degradation pathway, while accumulation of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed. These results show that R. erythropolis DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone. This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate. In the presence of coenzyme A and ATP this acid is converted further, and this finding, together with the high levels of isocitrate lyase activity in extracts of limonene-grown cells, suggests that further degradation takes place via the beta-oxidation pathway.     

Rhodococcus erythropolis DCL14 Contains a Novel Degradation Pathway for Limonene

Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample. This organism was identified as a strain of Rhodococcus erythropolis by chemotaxonomic and genetic studies. R. erythropolis DCL14 also assimilated the terpenes limonene-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (−)-menthol, while perillyl alcohol was not utilized as a carbon and energy source. Induction tests with cells grown on limonene revealed that the oxygen consumption rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of R. erythropolis DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1,2-monooxygenase activity, a cofactor-independent limonene-1,2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation studies showed that (1S,2S,4R)-limonene-1,2-diol, (1S,4R)-1-hydroxy-2-oxolimonene, and (3R)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)-limonene degradation pathway. The opposite enantiomers [(1R,2R,4S)-limonene-1,2-diol, (1R,4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxoheptanoate] were found in the (4S)-limonene degradation pathway, while accumulation of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed. These results show that R. erythropolis DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone. This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate. In the presence of coenzyme A and ATP this acid is converted further, and this finding, together with the high levels of isocitrate lyase activity in extracts of limonene-grown cells, suggests that further degradation takes place via the β-oxidation pathway.     

Reconstruction of a genome-scale metabolic network of Rhodococcus erythropolis for desulfurization studies

The remarkable catabolic diversity of Rhodococcus erythropolis makes it an interesting organism for bioremediation and fuel desulfurization. However, a model that can describe and explain the combined influence of various intracellular metabolic activities on its desulfurizing capabilities is missing from the literature. Such a model can greatly aid the development of R. erythropolis as an effective desulfurizing biocatalyst. This work reports the reconstruction of the first genome-scale metabolic model for R. erythropolis using the available genomic, experimental, and biochemical information. We have validated our in silico model by successfully predicting cell growth results and explaining several experimental observations in the literature on biodesulfurization using dibenzothiophene. We report several in silico experiments and flux balance analyses to propose minimal media, determine gene and reaction essentiality, and compare effectiveness of carbon, nitrogen, and sulfur sources. We demonstrate the usefulness of our model by studying a few in silico mutants of R. erythropolis for improved biodesulfurization, and comparing the desulfurization abilities of R. erythropolis with an in silico mutant of E. coli.     

Intensification of surfactant synthesis in Rhodococcus erythropolis EK-1 cultivated on hexadecane

Activity of key enzymes of n-alkane metabolism was determined in cells of Rhodococcus erythropolis EK-1, a surfactant producer grown on n-hexadecane. Potassium cations were found to inhibit alkane hydroxylase and NADP(+)-dependent aldehyde dehydrogenase, while sodium cations were found to activate these enzymes. Decreased potassium concentration (to 1 mM), increased sodium concentration (to 35 mM), and addition of 36 micromol/l Fe(II), required for alkane hydroxylase activity, resulted in increased activity of the enzymes of n-hexadecane metabolism and in a fourfold increase of surfactant synthesis. A 1.5-1.7-fold increase in surfactant concentration after addition of 0.2% fumarate (gluconeogenesis precursor) and 0.1% citrate (lipid synthesis regulator) to the medium with n-hexadecane results from enhanced synthesis of trehalose mycolates, as evidenced by a 3-5-fold increase in phosphoenolpyruvate synthetase and trehalose phosphate synthase, respectively.     

Production of a Bioflocculant by Rhodococcus erythropolis S-1 Grown on Alcohols

Rhodococcus erythropolis strain S-1, which was isolated from soil, produces a bioflocculant. We have found that alcohols are useful carbon sources for its flocculant production. Ethanol was best for flocculant production and culture time. The bioflocculant produced on ethanol medium flocculated a wide range of suspended soils, alkaline and acid.     

Adaptation of Rhodococcus erythropolis cells to high concentrations of toluene

Cells of Rhodococcus erythropolis DCL14 were adapted to increasing toluene concentrations in a mechanically stirred reactor. When the initial non-adapted cells were placed in contact with toluene, only 10.5% of cells remained viable after 1 h in the presence of 20% (v/v) toluene, while 8.6% of cells were viable after 28 h in the presence of an organic phase containing 80% (v/v) toluene in n-dodecane. Cell adaptation was studied by following the toluene consumption rate, the viability of the cell population, and the composition of the bacteria cellular membrane in the presence of increasing concentrations of toluene in the reactor. A maximum toluene concentration of 4.9 M, which corresponds to 52.4% (v/v) toluene in the organic phase, was achieved, toluene being consumed at 10.7 mg/(h mg protein). The adapted cells showed a substantially increased resistance to 50% ethanol and to concentrations of Betadine and Micropur tablets currently used in water purification, when compared to non-adapted cells.