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PubMed2001

Degradation of 1,3-dichloropropene by a soil bacterial consortium and Rhodococcus sp. AS2C isolated from the consortium.

A bacterial consortium capable of degrading the fumigant 1,3-D ((Z)- and (E)- 1,3-dichloropropene) was enriched from an enhanced soil. This mixed culture degraded (Z)- and (E)-1,3-D only in the presence of a suitable biodegradable organic substrate, such as tryptone, tryptophan, or alanine. After 8 months of subculturing at 2- to 3-week intervals, a strain of Rhodococcus sp. (AS2C) that was capable of degrading 1,3-D cometabolically in the presence of a suitable second substrate was isolated. (Z)-3-chloroallyl alcohol (3-CAA) and (Z)-3-chloroacrylic acid (3-CAAC), and (E)-3-CAA and (E)-3-CAAC were the metabolites of (Z)- and (E)- 1,3-D, respectively. (E)- 1,3-D was degraded faster than (Z)- 1,3-D by the strain AS2C and the consortium. AS2C also degraded (E)-3-CAA faster than (Z)-3-CAA. Isomerization of (E)- 1,3-D to (Z)- 1,3-D or the (Z) form to the (E) form did not occur.

Allyl Compounds↗

PubMed2001

Physiology, biochemistry and taxonomy of deep-sea nitrile metabolising Rhodococcus strains.

A collection of nitrile-hydrolysing rhodococci was isolated from sediments sampled from a range of deep coastal, and abyssal and hadal trench sites in the NW Pacific Ocean, as part of our programme on the diversity of marine actinomycetes. Nitrile-hydrolysing strains were obtained by batch enrichments on nitrile substrates with or without dispersion and differential centrifugation pre-treatment of sediments, and were recovered from all of the depths sampled (approximately 1100-6500 m). Two isolates obtained from the Ryukyu (5425 m) and Japan (6475 m) Trenches, and identified as strains of Rhodococcus erythropolis, were chosen for detailed study. Both of the deep-sea isolates grew at in situ temperature (4 degrees C), salinities (0-4% NaCl) and pressures (40-60 MPa), results that suggest, but do not prove, that they may be indigenous marine bacteria. However, the absence of culturable Thermoactinomyces points to little or no run off of terrestrial microbiota into these particular trench sediments. Nitrile-hydrolysis by these rhodococci was catalysed by a nitrile hydratase-amidase system. The hydratase accommodated aliphatic, aromatic and dinitrile substrates, and enabled growth to occur on a much wider range of nitriles than the only other reported marine nitrile-hydrolysing R. erythropolis which was isolated from coastal sediments. Also unlike the latter strain, the nitrile hydratases of the deep-sea rhodococci were constitutive. The possession of novel growth and enzyme activities on nitriles by these deep-sea R. erythropolis strains recommends their further development as industrial biocatalysts.

DNA, Ribosomal↗

PubMed2001

Isobutylidenediurea degradation by Rhodococcus erythropolis.

A new enzyme (isobutylidenediurea amidinohydrolase) catalyzing the hydrolysis of isobutylidenediurea (a condensation product of urea and isobutyraldehyde widely used as a slow-release nitrogeneous fertilizer) was characterized from a strain of Rhodococcus erythropolis. The enzyme was purified 1,250-fold to apparent homogeneity and shown to hydrolyze the fertilizer to urea and isobutyraldehyde at a molar ratio of 2: 1. No activity was observed with ureido- or other structurally related compounds. Its molecular mass was determined by native polyacrylamide gelelectrophoresis and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry to be 15 kDa (+/-2 kDa) and 16.4 kDa, respectively. Growth of the bacterium in the presence of isobutylidenediurea led to an increased expression of the constitutively synthetized enzyme.

Biodegradation, Environmental↗

PubMed2002

Biodegradation of plasticizers by Rhodococcus rhodochrous.

Rhodococcus rhodochrous was grown in the presence of one of three plasticizers: bis 2-ethylhexyl adipate (BEHA), dioctyl phthalate (DOP) or dioctyl terephthalate (DOTP). None of the plasticizers were degraded unless another carbon source, such as hexadecane, was also present. When R. rhodochrous was grown with hexadecane as a co-substrate, BEHA was completely degraded and the DOP was degraded slightly. About half of the DOTP was degraded, if hexadecane were present. In all of these growth studies, the toxicity of the media, which was assessed using the Microtox assay, increased as the organism degraded the plasticizer. In each case, there was an accumulation of one or two intermediates in the growth medium as the toxicity increased. One of these was identified as 2-ethylhexanoic acid and it was observed for all three plasticizers. Its concentration increased until degradation of the plasticizers had stopped and it was always present at the end of the fermentation. The other intermediate was identified as 2-ethylhexanol and this was only observed for growth in the presence of BEHA. The alcohol was observed early in the growth studies with BEHA and had disappeared by the end of the experiment. Both the 2-ethylhexanol and 2-ethylhexanoic acid were shown to be toxic and their presence explained the increase of toxicity as the fermentations proceeded. The appearance of these intermediates was consistent with similar degradation mechanisms for all three plasticizers involving hydrolysis of the ester bonds followed by oxidation of the released alcohol.

Alkanes↗

PubMed2003

Evidence of cytochrome P450-catalyzed cleavage of the ether bond of phenoxybutyrate herbicides in Rhodococcus erythropolis K2-3.

Bacterial strain Rhodococcus erythropolis K2-3 can cleave the ether bond of the phenoxybutyrate herbicides, i.e., 4-(2,4-dichlorophenoxy)butyrate (2,4-DB) and 4-(4-chloro-2-methylphenoxy)butyrate (MCPB), by an enzyme system that is constitutively expressed. The enzyme(s) involved were investigated in this study. The rate of disappearance of 2,4-DB determined in a whole cell assay amounted to 0.6 mmol/h x g(dry mass). Carbon monoxide difference spectra of dithionite-reduced whole cells and crude cell extracts suggested that strain K2-3 contains a soluble cytochrome P450 (P450), named P450(PB-1). The addition of various phenoxybutyrate substrates to crude cell extracts resulted in typical difference spectra following the type I pattern of substrate binding with P450. The rate of 2,4-DB cleavage was reduced by inhibitors of P450: 5 mM metyrapone and carbon monoxide at a CO/O2 ratio of 10 reduced the activity by about 20%, and 70%, respectively. The ether cleaving activity completely disappeared after disruption of the cells and could not be detected in crude extracts. To elucidate the enzymatic basis of this reaction, P450 was partially purified. With the resulting enzyme preparation, 2,4-DB cleavage activity was re-established, becoming measurable after the addition of either phenazine methosulfate or ferredoxin and ferredoxin/NADP oxidoreductase from spinach. We detected no activities attributable to alpha-ketoglutarate-dependent dioxygenase or NAD(P)H-dependent monooxygenase. These results collectively indicate that cleavage of the ether bond of phenoxybutyrate herbicides is catalyzed by P450-mediated activity in this strain. One of the products derived from this reaction is dichlorophenol, and comparative chromatographic analyses suggest that the other product is a C4-carbonic acid, most likely succinic semialdehyde/succinate.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2003

Enhanced desulfurization in a transposon-mutant strain of Rhodococcus erythropolis.

The dsz desulfurization gene cluster from Rhodococcus erythropolis strain KA2-5-1 was transferred into R. erythropolis strain MC1109, unable to desulfurize light gas oil (LGO), using a transposon-transposase complex. As a result, two recombinant strains, named MC0203 and MC0122, were isolated. Resting cells of strain MC0203 decreased the sulfur concentration of LGO from 120 mg l(-1) to 70 mg l(-1) in 2 h. The LGO-desulfurization activity of strain MC0203 was about twice that of strain MC0122 and KA2-5-1. The 10-methyl fatty acids of strain MC0203 were about 28%-41% that of strain MC1109. It is likely that strain MC0203 had a mutation involving alkylenation or methylation of delta9-unsaturated fatty acids caused by the transposon inserted in the chromosome, which increased the fluidity of cell membranes and enhanced the desulfurization activity.

Biotransformation↗

PubMed2004

Rhodococcus pyridinovorans MW3, a bacterium producing a nitrile hydratase.

Rhodococcus pyridinovorans MW3 was isolated from an arable land of manioc from the Congo for its ability to transform acrylonitrile to acrylamide. This strain contains a cobalt nitrile hydratase (NHase) showing high sequence homology with NHases so far described. The specific NHase activity was 97 U mg(-1) dry wt. NHase production by R. pyridinovorans MW3 was urea and Co-dependent. The NHase was active for acrylamide up to 60% (w/v) indicating its potential for acrylamide production.

Acrylamide↗

PubMed2002

Rhodococcus equi pulmonary infection in a pancreas-alone transplant recipient: consequence of intense immunosuppression.

We report the case of a pancreas-alone transplant recipient who developed Rhodococcus equi pneumonia after receiving multiple courses of antilymphocyte therapy for the treatment of recurrent acute pancreas allograft rejection. We also review and discuss the diagnosis, clinical course, and treatment of 18 cases of R. equi infection reported in solid organ transplant recipients. The lung is the most common primary site of infection, but R. equi infection is difficult to diagnose because of the pleomorphic, gram-positive, and partially acid-fast nature of the organism. Treatment usually involves a combination of antibiotics including rifampin, macrolides, vancomycin, and ciprofloxacin. The optimal duration of therapy is unknown, but relapse is common if the duration of treatment is less than 14 days. The duration of therapy should be guided by clinical recovery, culture results, and radiographic findings. Monitoring levels of immunosuppressive agents-such as tacrolimus and cyclosporine-is needed in order to avoid clinically significant drug interactions with rifampin or the macrolides when these agents are used in order to treat R. equi infection in the transplant population.

Actinomycetales Infections↗

PubMed2003

Cavitary pneumonia due to Rhodococcus equi in a heart transplant recipient.

Rhodococcus equi is an uncommon human pathogen that usually affects immunocompromised patients. We present a case of a 68-year-old male heart transplant recipient, who developed rhodococcal pneumonia with secondary bacteremia 10 months post-transplant. The patient was a retired carpenter who was involved in breeding of horses. He responded completely to the treatment with vancomycin and imipenem/cilastin, followed by oral ciprofloxacin and minocycline for total treatment duration of 5 months. This case highlights the association between an animal exposure and infection with a unique opportunistic pathogen.

Actinomycetales Infections↗

PubMed2003

Highly enantioselective synthesis of alpha,alpha-disubstituted malonamic acids through asymmetric hydrolysis of dinitriles with Rhodococcus sp. CGMCC 0497.

Highly enantioselective hydrolysis of alpha,alpha-disubstituted malononitriles by the strain Rhodococcus sp. CGMCC 0497 expressing both nitrile hydratase and amidase activity to give (R)-alpha,alpha-disubstituted malonamic acids which could be converted to valuable (R)- or (S)-alpha-alkylated amino acids are reported and the yields of the products are improved remarkably at a lower reaction temperature.

Amidohydrolases↗

PubMed2000

Purification and characterization of a Baeyer-Villiger mono-oxygenase from Rhodococcus erythropolis DCL14 involved in three different monocyclic monoterpene degradation pathways.

A Baeyer-Villiger mono-oxygenase (BVMO), catalysing the NADPH- and oxygen-dependent oxidation of the monocyclic monoterpene ketones 1-hydroxy-2-oxolimonene, dihydrocarvone and menthone, was purified to homogeneity from Rhodococcus erythropolis DCL14. Monocyclic monoterpene ketone mono-oxygenase (MMKMO) is a monomeric enzyme of molecular mass 60 kDa. It contains 1 mol of FAD/monomer as the prosthetic group. The N-terminal amino acid sequence showed homology with many other NADPH-dependent and FAD-containing (Type 1) BVMOs. Maximal enzyme activity was measured at pH 9 and 35 degrees C. MMKMO has a broad substrate specificity, catalysing the lactonization of a large number of monocyclic monoterpene ketones and substituted cyclohexanones. The natural substrates 1-hydroxy-2-oxolimonene, dihydrocarvone and menthone were converted stoichiometrically into 3-isopropenyl-6-oxoheptanoate (the spontaneous rearrangement product of the lactone formed by MMKMO), 4-isopropenyl-7-methyl-2-oxo-oxepanone and 7-isopropyl-4-methyl-2-oxo-oxepanone respectively. The MMKMO-catalysed conversion of iso-dihydrocarvone showed an opposite regioselectivity to that of dihydrocarvone; in this case, 6-isopropenyl-3-methyl-2-oxo-oxepanone was formed as the product. MMKMO converted all enantiomers of the natural substrates with almost equal efficiency. MMKMO is involved in the conversion of the monocyclic monoterpene ketone intermediates formed in the degradation pathways of all stereoisomers of three different monocyclic monoterpenes, i.e. limonene, (dihydro)carveol and menthol.

Aldehydes↗

PubMed2002

Growth and production of cholesterol oxidase by alginate-immobilized cells of Rhodococcus equi No. 23.

Rhodococcus equi No. 23 was immobilized in calcium alginate. No detrimental effect on the viability of the test organism was observed during the immobilization procedure. Approx. 98% of the cell population originally present in the alginate solution were immobilized in the gel beads. When the cells of an equal volume of the culture, obtained respectively at exponential phase (12 h preculture), late-exponential phase (20 h preculture) or stationary phase (36 h preculture) were immobilized, the gel beads prepared with the stationary-phase culture were found to contain the highest cell population [about 10(8) colony-forming units (CFU)/g of beads]. In addition, gel beads, prepared with late-exponential-phase culture, exhibited the highest production of cholesterol oxidase (CholOx) after 48 h of incubation. Increasing the bead mass from 3.5 to 14.0 g/100 ml of medium increased CholOx production. However, further increasing the bead mass resulted in a reduction of CholOx production. Furthermore, on the basis of a similar initial cell population, the alginate-immobilized cells of R. equi No. 23 produced a significantly higher amount of CholOx (P<0.05) than did the free cells.

Alginates↗

PubMed2002

Enhancement of enzyme activity and enantioselectivity via cultivation in nitrile metabolism by Rhodococcus sp. CGMCC 0497.

Racemic 2-phenylpropionitrile was resolved enantioselectively by nitrile-converting enzymes in cells of Rhodococcus sp. CGMCC 0497 to S-(+)-2-phenylpropionic acid and R-(-)-2-phenylpropionamide. By optimization of the culture conditions, great enhancement of enzyme activity and enantioselectivity was achieved. Furthermore, the relationship between cell-growth periodicity and enzyme accumulation was studied; the addition of inducer was delayed by 1 day and the reaction was further improved. This unusual strategy has almost never been reported with other nitrile-converting strains. The resulting culture broth, containing methacrylamide as the inducer, beef extract as the nitrogen source and glucose as the carbon source, with methacrylamide added 24 h later, seemed to be most suitable. S-(+)-2-Phenylpropionic acid and R-(-)-2-phenylpropionamide were produced with yields of 48% (enantiomeric excess, 96%) and 42% (enantiomeric excess, 97%) respectively with no nitrile left after 3 h, or with yields of 52% and 39% (enantiomeric excess, 93% and 99%) respectively after 6 h.

Acetonitriles↗

PubMed2004

Biotransformation of geraniol by Rhodococcus sp. strain GR3.

Microbial degradation of geraniol, a natural monoterpene alcohol, was studied using a Rhodococcus sp. strain GR3 isolated from soil. The bioconversion product was identified as geranic acid [(2 E )-3,7-dimethylocta-2,6-dienoic acid] and its structure was established by (1)H-NMR, Fourier-transform IR spectrometry and GC-MS. The optimum temperature for this bioconversion was found to be 30 degrees C, and the reaction proceeds to a saturation with a time constant of 12.5 h. No appreciable degradation of product was observed using this bacterium.

Acyclic Monoterpenes↗

PubMed1996

Reciprocal action of interferon-gamma and interleukin-4 promotes granulomatous inflammation induced by Rhodococcus aurantiacus in mice.

An intravenous injection of Rhodococcus aurantiacus to mice causes granulomatous inflammation dependent on endogenous interferon-gamma (IFN-gamma). The present study examined the role of endogenous interleukin-4 (IL-4) on granulomatous inflammation. Endogenous IL-4 in the spleen extracts was not detected during the phase of granuloma formation by enzyme-linked immunosorbent assay (ELISA). However, IL-4 protein level was elevated during the phase of granuloma regression. IL-4 mRNA expression in the livers and spleens was also elevated during the phase of granuloma regression. In addition, IL-4 levels during the phase of granuloma formation were increased by treatment with anti-IFN-gamma monoclonal antibody (mAb), suggesting that endogenous IFN-gamma might inhibit IL-4 production during the phase of granuloma formation. Administration of anti-IL-4 mAb on weeks 3 and 4 after the inoculation inhibited the regression of granulomas and augumented IFN-gamma level at 5 weeks. Endogenous IFN-gamma was produced by CD4+ T cells during the phase of granuloma regression and endogenous IL-4 was produced by both CD4+ and CD8+ T cells. These findings suggest that during the phase of granuloma formation endogenous IL-4 might be inhibited by IFN-gamma, while during the phase of granuloma regression endogenous IL-4 might play a crucial role in the reduction of granulomas and IFN-gamma production.

Actinomycetales Infections↗

PubMed1999

Tumour necrosis factor and interferon-gamma are required in host resistance against virulent Rhodococcus equi infection in mice: cytokine production depends on the virulence levels of R. equi.

Rhodococcus equi is a facultative intracellular bacterial pathogen that causes pneumonia in foals and immunosuppressed humans. There are at least three virulence levels of R. equi and these pathogenicities are associated, in mice, with the presence of virulence plasmids. This study focused on cytokine secretion, in mice, in the course of a primary infection with sublethal doses of R. equi strains of different virulence levels (virulent, intermediately virulent and avirulent). Tumour necrosis factor (TNF) and interferon-gamma (IFN-gamma), but not interleukin-4 (IL-4) and interleukin-10 (IL-10), were induced endogenously in mice in relation to the multiplication and clearance of virulent and intermediately virulent strains of R. equi. These cytokines were not detected in mice infected with avirulent R. equi. Deaths occurred among mice treated with monoclonal antibodies (mAbs) against either TNF or IFN-gamma prior to sublethal dose infection with virulent and intermediately virulent strains of R. equi, but not with avirulent R. equi. These results suggested that cytokine production depended largely on the virulence levels of R. equi: TNF and IFN-gamma were required early during infection with virulent R. equi to limit replication and clearance of bacteria within the organs, but they were not necessary for limiting infection with avirulent R. equi.

Actinomycetales Infections↗

PubMed1999

Movement disorders in encephalitis induced by Rhodococcus aurantiacus infection relieved by the administration of L-dopa and anti-T-cell antibodies.

Mice injected with Rhodococcus aurantiacus by the intravenous (i.v.) route show neurological disorders, hemiparesis, vertical headshake and turn-round gait after day 7 postinfection (p.i.). Neurological symptoms caused by i.v. inoculation of R. aurantiacus were relieved by treatment with levodopa (l-dopa). R. aurantiacus was isolated from the brain and was found to be completely eliminated at day 7 p. i. Focal encephalitis was mainly observed in the brain stem, and T cells could be isolated from the brain after day 7 p.i. Administration of both an anti-CD4 monoclonal antibody (mAb) and an anti-CD8 mAb suppressed neurological symptoms. These results suggest that R. aurantiacus induces movement disorders in mice, and that the symptoms are mediated by T cells infiltrating the brain, rather than directly by the bacterium.

Actinomycetales Infections↗

PubMed1999

Transcriptional analysis of the nitrile-degrading operon from Rhodococcus sp. ACV2 and high level production of recombinant amidase with an Escherichia coli-T7 expression system.

Northern blotting analysis with RNA probes derived from amidase and nitrile hydratase genes from Rhodococcus sp. ACV2 revealed that both genes are part of the same operon. RNase protection mapping and sequence analysis indicated that the operon is probably under the control of a sigma 70-like promoter located upstream from the amidase gene. Plasmids were constructed with the cloned genes under tac and lac promoter control. Expression of amdA was demonstrated in Escherichia coli. In another construction, the amdA gene was inserted under the control of the bacteriophage T7 promoter. Large amounts of recombinant amidase (at least 20% of total proteins) in a soluble and active form were obtained with the E. coli-T7 expression system by lowering the growth temperature to 29 degrees C, without IPTG induction. The ratio of amidase activity of strain ACV2 to E. coli was approximately 1:3. Purification of the recombinant amidase was carried out in one chromatographic step, giving an enzyme preparation that could be used directly in a biotechnological process.

Amidohydrolases↗