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PubMed1997

Development of a Rhodococcus equi-Escherichia coli plasmid shuttle vector.

Isolates of Rhodococcus equi from pneumonic foals possess an 85- or 90-kb virulence-associated plasmid. A prominent, thermoregulated surface antigen, VapA, encoded by these plasmids is thought to be important in virulence. A 135-kb fragment containing the origin of replication of R. equi strain 103 virulence-associated plasmid (pOTS) was identified, sequenced, and its location identified. A simple R. equi-Escherichia coli shuttle plasmid (pRE-1) derived from the E. coli plasmid pACYC177 and the pOTS ori was developed. The plasmid transformed readily and was stable in either host and expressed kanamycin resistance but not beta-lactamase in R. equi. An improved 5.9-kb vector, pRE-7, was developed from pRE-1 and pBluescript. Subcloning of vapA into the multiple cloning site of the beta-galactosidase gene of pRE-7 resulted in weak expression of the gene both in E. coli and R. equi. The shuttle vector may be useful in examining regulation of virulence gene expression in R. equi.

Bacterial Proteins↗

PubMed1996

Biodegradation of 4-nitroanisole by two Rhodococcus spp.

Two Rhodococcus strains, R. opacus strain AS2 and R. erythropolis strain AS3, that were able to use 4-nitroanisole as the sole source of carbon and energy, were isolated from environmental samples. The first step of the degradation involved the O-demethylation of 4-nitroanisole to 4-nitrophenol which accumulated transiently in the medium during growth. Oxygen uptake experiments indicated the transformation of 4-nitrophenol to 4-nitrocatechol and 1,2,4-trihydroxybenzene prior to ring cleavage and then subsequent mineralization. The nitro group was removed as nitrite, which accumulated in the medium in stoichiometric amounts. In R. opacus strain AS2 small amounts of hydroquinone were produced by a side reaction, but were not further degraded.

Anisoles↗

PubMed1991

Dechlorination of pentachlorophenol by membrane bound enzymes of Rhodococcus chlorophenolicus PCP-I.

Dechlorination (para-hydroxylation) of pentachlorophenol (PCP) and tetrachloro-para-hydroquinone (TeCH) and O-methylation of TeCH were demonstrated in cell extracts of Rhodococcus chlorophenolicus PCP-I. PCP para-hydroxylating activity was membrane bound, whereas TeCH dechlorinating enzyme was soluble. The PCP para-hydroxylating enzyme was solubilized by Triton X-100 and the requirement for both FAD and NADPH was shown. The dechlorinating activities were inducible in contrast to the constitutive TeCH O-methylating activity. The PCP para-hydroxylation was inhibited by its product TeCH, by anoxic conditions, and by different inhibitors of P450. Participation of this cytochrome in the PCP hydroxylation was confirmed by the appearance of a carbon monoxide dependent peak of absorbance at 457 nm in the membrane fraction prepared from PCP degrading cells.

Biodegradation, Environmental↗

PubMed1992

Distribution and antimicrobial susceptibility of Rhodococcus equi from clinical specimens.

Rhodococcus equi, an unusual gram positive aerobic actinomycete, was first described as a respiratory pathogen of livestock in 1923. Reports of human clinical illness have emphasized R. equi as a cause of invasive pulmonary infection in severely immunocompromised patients and, recently, have implicated it as a cause of pneumonia, bacteremia and disseminated infection in HIV-infected patients. To determine the distribution of R. equi we evaluated 107 isolates referred to the Centers for Disease Control (CDC) during the period January 1973 through December 1990. The sites of these 107 isolates (101 patient and 6 animal isolates) were: blood (32 isolates), sputum (30), lung tissue (13) and other site (32). Before 1983, when the first R. equi isolate from an HIV-infected patient was received, CDC received a total of 52 patient isolates. In addition, during this 10 year period, R. equi isolates were received from more than one site from only one patient. However, during the two year period 1989-1990, we identified 8 patients with underlying HIV infection and R. equi pneumonia who accounted for 29 of 35 (83%) R. equi patient isolates; 6 of these patients also had bacteremia and three died with disseminated R. equi infection. No isolates were resistant to amoxicillin-clavulanate, ampicillin-sulbactam, gentamicin or imipenem, and few (less than 5%) isolates were resistant to erythromycin, rifampin, tetracycline, and trimethoprim-sulfamethoxazole. These results suggest that HIV-infected patients, in particular, are predisposed to develop invasive pulmonary, fatal disseminated R. equi infection (or both), and appropriate antimicrobial susceptibility testing of clinical isolates may improve the effectiveness of therapy of R. equi-infected patients.

Actinomycetales Infections↗

PubMed1993

Production of S-(+)-2-phenylpropionic acid from (R,S)-2-phenylpropionitrile by the combination of nitrile hydratase and stereoselective amidase in Rhodococcus equi TG328.

A new soil isolate, tentatively identified as Rhodococcus equi TG328, was found to be effective in the production of S-(+)-2-phenylpropionic acid from (R,S)-2-phenylpropionitrile. The conversion is catalysed by two enzymes. First, a nitrile hydratase converts the (R,S)-nitrile to (R,S)-2-phenylpropionamide. Second, a stereoselective amidase converts the S-(+)-amide to S-(+)-2-phenylpropionic acid. Conditions for optimal enzyme production and accumulation of S-(+)-2-phenylpropionic acid by resting cells were studied. The reaction of resting cells for 30 h at 10 degrees C with (R,S)-2-phenylpropionitrile resulted in the production of 100 g of S-(+)-2-phenylpropionic acid per litre of reaction mixture. The enantiometric excess of the purified S-(+)-2-phenylpropionic acid was 99.4%. The amount of S-(+)-2-phenylpropionic acid accumulated was enhanced by lower reaction temperatures. In addition, unreacted R-(-)-2-phenylpropionamide with 99.0% enantiometric excess was isolated.

Acetonitriles↗

PubMed1991

Degradation of dioxane, tetrahydrofuran and other cyclic ethers by an environmental Rhodococcus strain.

By enrichment and isolation techniques bacterial strains with the capacity to grow on aliphatic cyclic ethers (dioxane, tetrahydrofuran, 1,3-dioxolane) have been isolated. Six strains that degrade tetrahydrofuran were classified as belonging to the genus Rhodococcus. One of two strains that degrade dioxane instead of or in combination with tetrahydrofuran was further characterized and a hypothetical catabolic pathway comprising an initial 2-hydroxylation and several oxidation steps is postulated.

Biodegradation, Environmental↗

PubMed1990

Aryl acylamidase from Rhodococcus erythropolis NCIB 12273.

A Rhodococcus erythropolis strain was isolated from soil on the basis of its ability to use acetaminophen as the sole source of both carbon and energy for growth. When grown in a complex medium containing an anilide inducer compound, the bacterium exhibited aryl acylamidase (EC 3.5.1.13) activity. This activity was not subject to carbon or nitrogen repression by the growth medium constituents as the enzyme was present throughout the exponential growth phase. The anilide was converted to the corresponding aniline, which was not further degraded. The enzyme was partially purified by a variety of methods including a batch ion exchange procedure, column ion exchange chromatography and hydrophobic interaction chromatography. The enzyme had a maximum activity at around pH 8.0 and had a Km for acetaminophen of 0.11 mM. Electrochemical assays of aryl acylamidase activity are described. The enzyme is suitable for use as a reagent in the clinical diagnostic measurement of acetaminophen.

Acetaminophen↗

PubMed1996

Radiological findings in nine AIDS patients with Rhodococcus equi pneumonia.

Rhodococcus equi (R. equi) infections have been incidentally reported as a cause of pulmonary infection in severely immunocompromised hosts, including AIDS patients. Our purpose is to describe the radiological findings in nine AIDS patients with R. equi pneumonia assessed by bronchoalveolar lavage (BAL), biopsies, cultures of sputum, and hemocultures. All patients were examined by chest radiographs and contrast-medium-enhanced chest CT. Dense pulmonary consolidations with or without cavitations accounted for the most striking radiological patterns. Chest CT also revealed six mediastinal involvements, strongly mimicking a lymphoma. Two of them had multiple bilateral pulmonary nodular opacities. Pleural effusion was not identified. Although intensive therapies were administered, seven among nine patients died within few months. In an AIDS patient living in a rural area or exposed to horses and presenting these radiological patterns, the possibility of R. equi pneumonia should be considered in the differential diagnosis along with other infectious diseases or lymphomas.

AIDS-Related Opportunistic Infections↗

PubMed1991

Catabolism of 1,3-dinitrobenzene by Rhodococcus sp. QT-1.

The 1,3-dinitrobenzene-degrading Rhodococcus strain QT-1 was isolated under nitrogen limiting conditions from contaminated soil samples. Experimental data indicate tha 1,3-dinitrobenzene is metabolized via 4-nitrocatechol. Both compounds were oxidized by resting cells and nitro groups were completely eliminated as nitrite. Strain QT-1 utilizes both 1,3-dinitrobenzene and 4-nitrocatechol as source of nitrogen in the absence as well as in the presence of high amounts of ammonia. Growth on 4-nitrocatechol does not induce the enzyme(s) for the initial oxidation of 1,3-dinitrobenzene.

Biodegradation, Environmental↗

PubMed1988

Plasmid-borne resistance to arsenate, arsenite, cadmium, and chloramphenicol in a Rhodococcus species.

A primarily genetic approach was employed to obtain plasmids in Rhodococcus erythropolis ATCC 12674 which carried genes conferring increased resistance to sodium arsenate and arsenite, cadmium chloride, and chloramphenicol. The plasmids were large, migrating more slowly than chromosomal DNA in agarose gels, and were made up of resistance determinants from the host organism together with part of the genome of nocardiophage Q4. Purified plasmid was used to transform a suitable recipient to increased resistance to sodium arsenate, sodium arsenite, and cadmium chloride.

Arsenates↗

PubMed1990

Kinetic studies of phenol degradation by Rhodococcus sp. P1. I. Batch cultivation.

Rhodococcus sp. P1 utilizes phenol as the sole carbon and energy source via the beta-ketoadipate pathway. In batch cultivation, concentrations up to 2.8 g.l-1 phenol were degraded. The highest values for the specific growth rate of 0.32 h-1 were obtained at concentrations near 0.25 g.l-1. At higher concentrations, substrate inhibition was observed, characterized by increases in lag phase and decreasing growth rates. A mathematical expression was proposed to fit the kinetic pattern of phenol inhibition on the specific growth rate mu: [formula: see text] Nomenclature: K- Exponent of the inhibition function, Ks- Monod saturation constant, g.l-1, KI- Inhibition constant, g.l-1, S- Substrate concentration in culture broth, g.l-1, So- Initial substrate concentration, g.l-1, Y- Yield constant, g cell dry mass.g substrate-1, mu- Specific growth rate, h-1, mu max- Maximum growth rate, h-1.

Culture Media↗

PubMed1990

Kinetic studies of phenol degradation by Rhodococcus sp. P1. II. Continuous cultivation.

The degradation of phenol by Rhodococcus sp. P1 was studied in continuous culture systems. The organism could be adapted by slowly increasing concentration, step by step, up to 30.0 g.l-1 phenol in the influent. The degradation rate reached values of about 0.3 g.g dry mass-1.h-1. Large step increases in phenol concentration and addition of further substrates (e.g., catechol) were tolerated up to a certain concentration. With increasing dilution rate and increasing inlet phenol concentration the stability of the system decreased. Nomenclature: D--Dilution rate, h-1, Dc--Critical dilution rate, h-1, Dx--Yield, g dry mass.l-1.h-1, Ks--Monod saturation constant, g.l-1, S--Growth-limiting substrate concentration in culture broth, g.l-1, SR--Growth-limiting substrate concentration in feed, g.l-1, mean--Biomass concentration in culture broth, g.l-1, YS--Yield constant, g cell dry mass.g substrate-1, mu--Specific growth rate, h-1, mu max--Maximum growth rate, h-1.

Culture Media↗

PubMed1988

Bacterial metabolism of side chain fluorinated aromatics: cometabolism of 3-trifluoromethyl(TFM)-benzoate by Pseudomonas putida (arvilla) mt-2 and Rhodococcus rubropertinctus N657.

The TOL plasmid-encoded enzymes of the methylbenzoate pathway in Pseudomonas putida mt-2 cometabolized 3-trifluoromethyl (TFM)-benzoate. Two products, 3-TFM-1,2-dihydroxy-2-hydrobenzoate (3-TFM-DHB) and 2-hydroxy-6-oxo-7,7,7-trifluoro-hepta-2,4-dienoate (7-TFHOD) were identified chemically and by spectroscopic properties. TFM-substituted analogues of the metabolites of the methylbenzoate pathway were generally converted at drastically reduced rates. The catechol-2,3-dioxygenase from Pseudomonas putida showed moderate turnover rates with 3-TFM-catechol. The catechol-1,2-dioxygenase of Rhodococcus rubropertinctus N657 was totally inhibited by 3-TFM-catechol and did not cleave this substrate. Hammett-type analysis showed the catechol-1,2-dioxygenase reaction to be strongly dependent on the electronic nature of the substituents. Electronegative substituents strongly inhibited catechol cleavage. The catechol-2,3-dioxygenase reaction, however, was only moderately sensitive to electronegative substituents.

Biodegradation, Environmental↗

PubMed1995

Metabolism of halohydroquinones in Rhodococcus chlorophenolicus PCP-1.

The actinomycete Rhodococcus chlorophenolicus PCP-1 metabolizes pentachlorophenol into ultimate inorganic end products via tetrachloro-p-hydroquinone. This intermediate was further dehalogenated in the cytoplasm requiring reductant in the cell free system. Tetrafluoro-p-hydroquinone and tetrabromo-p-hydroquinone were also dehalogenated. Chlorophenol analogs, thiol blocking agents and molecular oxygen inhibited the activity. The dehalogenating reactions led to 1,2,4-trihydroxybenzene, which was further metabolized into maleic acid.

Biodegradation, Environmental↗

PubMed1995

Dehalogenation of haloalkanes by Rhodococcus erythropolis Y2. The presence of an oxygenase-type dehalogenase activity complements that of an halidohydrolase activity.

Rhodococcus erythropolis Y2 produced two types of dehalogenase: a hydrolytic enzyme, that is an halidohydrolase, which was induced by C3 to C6 1-haloalkane substrates, and at least one oxygenase-type dehalogenase induced by C7 to C16 1-haloalkanes and n-alkanes. The oxygenase-type activity dehalogenated C4 to C18 1-chloroalkanes with an optimum activity towards 1-chlorotetradecane. The halidohydrolase catalysed the dehalogenation of a wide range of 1- and alpha,omega-disubstituted haloalkanes and alpha,omega-substituted haloalcohols. In resting cell suspensions of hexadecane-grown R. erythropolis Y2 the oxygenase-type dehalogenase had a specific activity of 12.9 mU (mg protein)-1 towards 1-chlorotetradecane (3.67 mU mg-1 towards 1-chlorobutane) whereas the halidohydrolase in 1-chlorobutane-grown batch cultures had a specific activity of 44 mU (mg protein)-1 towards 1-chlorobutane. The significance of the two dehalogenase systems in a single bacterial strain is discussed in terms of their contribution to the overall catabolic potential of the organism.

Alkanes↗

PubMed1995

Sizing of the Rhodococcus sp. R312 genome by pulsed-field gel electrophoresis. Localization of genes involved in nitrile degradation.

The two restriction enzymes AsnI and DraI were found to produce DNA fragment sizes that could be used for mapping the Rhodococcus sp. R312 (formerly Brevibacterium sp. R312) genome by pulsed-field gel electrophoresis. AsnI produced 24 fragments (4 to 727 kb) and DraI yielded 15 fragments (8.5 to 2400 kb). The fragment lengths in each digest were summed, indicating that the size of the chromosome ranged from 6.31 to 6.56 Mb, with a mean of 6.44 Mb. In addition, the wide-spectrum amidase gene (amiE) and the operon containing the enantiomer-selective amidase gene (amdA) and the nitrile hydratase structural gene (nthA, nthB) were localized on the AsnI and DraI fragments.

Amidohydrolases↗

PubMedDate not supplied

Osteomyelitis caused by Rhodococcus equi in a renal transplant recipient.

We report the first case of osteomyelitis due to Rhodococcus equi, which occurred in a renal transplant patient. Infection with this organism is rare and usually causes a distinct clinical syndrome resembling pulmonary tuberculosis. We investigated by time-kill curve analysis various antimicrobial combinations for in vitro efficacy. The literature is briefly reviewed, and aspects of diagnosis and therapy are discussed.

Actinomycetales Infections↗

PubMed1997

Improvement of lysine production by analog-sensitive and auxotroph mutants of the acetylene-utilizing bacterium Gordona bronchialis (Rhodococcus bronchialis).

An acetylene utilizing Gordona (Rhodococcus) bronchialis strain, screened for the production of fine chemicals, was found to be capable of producing small amounts of lysine. Attempts to produce amino-acid analog-resistant and/or sensitive mutants and auxotrophs of this strain with increased lysine production were made following UV-irradiation or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. The bacterium exhibited surprisingly high resistance levels to the aforementioned mutagens which is attributed to highly effective inborn-repair systems. Natural resistance to high levels of S-(2-aminoethyl)-L-cysteine (AEC) (2%) was observed, in contrast with D, L-aspartic acid hydroxamate (AAH), L-lysine hydroxamate (LHX) and beta-fluoropyruvate (FP). A variety of amino-acid analog-resistant (AAHr, LHXr) or analog-sensitive (FPs) mutants were produced following UV-irradiation or MNNG treatment. Similarly, a large number of auxotrophs (68) of different types were also obtained. From these, one FPs mono-auxotroph and two poly-auxotrophs (with at least one requirement for the aspartic acid family) showed an increased lysine production (approximately 1.8 g/L) comparable (4 g/L) to that found in other bacteria capable of utilizing long-chain hydrocarbons (1).

Acetylene↗