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PubMed1999

Purification and characterization of dibenzothiophene (DBT) sulfone monooxygenase, an enzyme involved in DBT desulfurization, from Rhodococcus erythropolis D-1.

Dibenzothiophene (DBT), a model of organic sulfur compound in petroleum, is microbially desulfurized to 2-hydroxybiphenyl by Rhodococcus erythropolis D-1. Three desulfurization (Dsz) enzymes--DszC, A, and B--and flavin reductase are involved in sulfur-specific DBT desulfurization. In this study, DszA was purified, characterized, and crystallized from R. erythropolis D-1. DszA, DBT sulfone monooxygenase, is the second enzyme in microbial DBT desulfurization metabolism and catalyzes the conversion of DBT sulfone to 2'-hydroxybiphenyl 2-sulfinic acid in the presence of flavin reductase with cleavage of the carbon-sulfur bond in the DBT skeleton. Using anion-exchange column chromatography, the four enzyme fractions responsible for DBT desulfurization were separated, and DszA was then purified to homogeneity. Polygonal crystals of DszA were observed within a week. DszA was found to have a molecular mass of 97 kDa and to consist of two subunits with identical masses of 50 kDa. The N-terminal amino acid sequence of the purified DszA completely coincided with the deduced amino acid sequence for dszA of R. erythropolis IGTS8 except for a methionine residue at the latter N-terminal. The optimal temperature and pH for DszA activity were 35 degrees C and about 7.5. The activity of the enzyme was inhibited by Mn2+, Ni2+, 2,2'-bipyridine, and 8-quinolinol, suggesting that a metal might be involved in its activity. DszA acted on not only DBT sulfone but also on dibenz[c,e][1,2]oxathiin 6-oxide and dibenz[c,e][1,2]oxathiin 6,6-dioxide. Dihydroxybiphenyl was formed from the latter two substrates.

Journal Article↗

PubMed2000

Increase in desulfurization activity of Rhodococcus erythropolis KA2-5-1 using ethanol feeding.

Rhodococcus erythropolis KA2-5-1 is one of the best strains for the desulfurization of dibenzothiophene (DBT) via a sulfur-specific pathway in which DBT is converted to the end product, 2-hydroxybiphenyl, by releasing sulfite via DBT-sulfone and 2-(2'-hydroxyphenyl) benzene sulphinate. The objective of this research is to develop a culture method in order to attain a high cell density with a high level of specific desulfurization activity. Compared with glucose or glycerol, ethanol was found to be a preferable carbon source for obtaining a high specific activity (SA) of desulfurization. When the amount of DBT fed was restricted by feeding 2.9 mg-DBT/g-ethanol solution, the maximum SA and final cell concentration were 135.5 (mmol-2HBP/kg-dry cell weight-h) and 37 (g-dry cell weight/l), respectively. On the other hand, when glucose or glycerol was used as a carbon source, the SA was lower than 50 (mM-2HBP/kg-dry cell weight-h) and the final cell concentration was also lower than 27 (g-dry cell weight/l). The activities of the desulfurization enzymes in R. erythropolis KA2-5-1 grown on ethanol were remarkably higher than when the strain was grown on glucose or glycerol. It was also suggested that NADH, which is produced by the biochemical reaction of NAD with ethanol catalyzed by alcohol dehydrogenase, might contribute to the conversion of FMN to FMNH2, which is a coenzyme for the activities of desulfurization enzymes.

Journal Article↗

PubMed2002

Diversity of 2,3-dihydroxybiphenyl dioxygenase genes in a strong PCB degrader, Rhodococcus sp. strain RHA1.

Two 2,3-dihydroxybiphenyl (23DHBP) dioxygenase genes, bphC1 and etbC involved in the degradation of polychlorinated biphenyl(s) (PCBs) have been isolated and characterized from a strong PCB degrader, Rhodococcus sp. RHA1. In this study, four new 23DHBP dioxygenase genes, designated as bphC2, bphC3, bphC4, and bphC5 were isolated from RHA1, and their nucleotide sequences were determined. Based on amino acid sequence similarities, all of the newly isolated bphC genes could be categorized into type I along with BphC1 and EtbC [Eltis, L.D. and Bolin, J.T., J. Bacteriol., 178, 5930-5937 (1996)]. Six bphC genes, including bphC1, etbC, and four new genes, were expressed in Escherichia coli to determine their substrate specificity. The activities of BphC2, BphC3, BphC4, and BphC5 were found to be specific to 23DHBP, while BphC1 and EtbC exhibited activities towards compounds other than 23DHBP, including catechol (CAT) and 3-methylcatechol (3MC). RNA slot blot hybridization analysis indicated that only bphC5 was transcribed among the newly isolated bphC in RHA1 cells grown on biphenyl and ethylbenzene. The nucleotide sequence of the flanking region of each bphC revealed a homolog of the 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPD) hydrolase gene, bphD, just upstream of bphC5. The bphC5 and putative bphD genes may constitute an operon and play a role in the degradation of biphenyl and PCBs together with bphC1 and etbC. In contrast, the bphC2, bphC3, and bphC4 genes may not be involved in biphenyl and PCB degradation.

Journal Article↗

PubMed2002

Enhancement and stabilization of desulfurization activity of Rhodococcus erythropolis KA2-5-1 by feeding ethanol and sulfur components.

We developed a fed-batch culture system fed with ethanol and restricted amounts of sulfur compounds to enhance and stabilize the desulfurizing activity in bacterial cells. In this system using dibenzothiophene (DBT) as the sole sulfur source, a desulfurizing bacterium Rhodococcus erythropolis KA2-5-1 cultivated with small amounts of sulfur showed stable desulfurizing activity and a low rate of growth. However, the cells cultured with excess amounts of sulfur showed unstable activity and a high growth rate. DBT had disadvantages as a sulfur source for cultivation because it is immiscible with water and toxic to cells. We then investigated water-soluble sulfur compounds for use as the sole sulfur source for the cultivation of R. erythropolis KA2-5-1 with desulfurizing activity, and found 2-aminoethanesulfonic acid to be the most effective. When 2-aminoethanesulfonic acid was used instead of DBT as the sole sulfur source in the fed-batch fermentation system, R. erythropolis KA2-5-1 showed the highest desulfurizing activity, 111 mmol of 2-HBP/kg-cells/h, a high growth rate (mu = 0.37/h) and a final cell concentration of 20.0 g-dry cells/l during 89 h of cultivation. The production levels of the desulfurizing enzymes in the bacterial cells cultivated with DBT or 2-aminoethanesulfonic acid were evaluated by immunoblot analysis with specific antisera, indicating that the same quantity of desulfurizing enzymes was expressed in both cases.

Journal Article↗

PubMed2002

Biodesulfurization of dibenzothiophene with Rhodococcus erythropolis ATCC 53968 and its mutant in an interface bioreactor.

An interface bioreactor was used in the biodesulfurization (BDS) of dibenzothiophene (DBT) with Rhodococcus erythropolis ATCC 53968. Although this strain could efficiently degrade DBT to 2-hydroxybiphenyl (2-HBP), the microbial film peeled off from the surface of the carrier (agar plate) to an organic phase (dodecane or tetradecane). To overcome this problem, a UV mutant, which strongly adhered to the carrier surface, was obtained by a new screening method. The best UV mutant (UM-021) could efficiently catalyze the BDS and the microbial film did not peel off from the surface of the carrier to tetradecane layer.

Journal Article↗

PubMed2004

Characteristics of Rhodococcus pyridinovorans PYJ-1 for the biodegradation of benzene, toluene, m-xylene (BTX), and their mixtures.

The degradation rates of benzene, toluene, and m-xylene (BTX) by Rhodococcus pyridinovorans PYJ-1, were highest at 30, 50, and 25 mg/l, respectively. The degradation rate was highest for toluene (0.106 mg/mg-protein x h) followed by benzene and m-xylene at the optimum pH and temperature of 7 and 32 degrees C, respectively. For BTX mixtures, toluene was the preferred substrate, but degradation of each BTX was competitively inhibited by other BTX compounds. The degradation rate of each component of in the BTX mixture decreased by 57-89% depending on the concentration (1-5 mg/l) of the component compared with that of the component as a single substrate.

Journal Article↗

PubMed2004

Constitutive synthesis, purification, and characterization of catechol 1,2-dioxygenase from the aniline-assimilating bacterium Rhodococcus sp. AN-22.

A catechol 1,2-dioxygenase (CD) was found, which was synthesized constitutively in the aniline-assimilating bacterium Rhodococcus sp. AN-22 grown on a medium without aniline, as well as on aniline medium. The bacterium synthesized CD in its cells grown on all the 21 non-aromatic substrates examined, including four natural media such as meat and yeast extracts, one sugar, six organic acids, and 10 amino acids as carbon, energy, and nitrogen sources. When the bacterium was incubated on a medium with D-glucose, L-malate, isoleucine, leucine, etc., it synthesized more CD than that in cells grown on aniline. Two CDs, which were prepared from cells grown on aniline and L-malate, were purified separately to homogeneity and characterized. The two enzymes were apparently identical in molecular and catalytic properties including molecular mass, optimal pH, stability to heating, and substrate specificity for catechol analogues. However, they differed in the substrate specificity and resistance to sulfhydryl and chelating agents from the inducible CDs produced by other aniline-assimilating bacteria reported previously.

Journal Article↗

PubMed1989

The association between weather, frenzied behaviour, percutaneous invasion by Strongyloides westeri larvae and Rhodococcus equi disease in foals.

Episodes of frenzy lasting approximately 30 minutes were observed among horses confined to enclosures surfaced with sand or soil. The probability of sighting these episodes increased by a factor of three when within 24 hours there was 0.2mm or more of rain, a maximum air temperature between 16.7 - 26.6 degrees C and a soil temperature of 16.3 - 23.9 degrees C at 30 cm. High egg counts of Strongyloides westeri appeared in faeces four to five days later and persisted for several days. Rhodococcus equi was recovered from all soil samples, and from the faeces of 76% of mares and 82% of foals. The youngest foal was five days old when the organism was isolated from rectal faeces. In contrast to the majority of reports, the lesions of R. equi in the foals were confined to limbs and peripheral lymph nodes. It is proposed that the percutaneous invasion of these foals by third stage larvae of S. westeri facilitated invasion of R. equi, and ubiquitous saprophytic opportunist pathogen.

Journal Article↗

PubMed1981

Differentiation between the genera Mycobacterium, Rhodococcus and Nocardia by susceptibility to 5-fluorouracil.

A test for susceptibility to 5-fluorouracil was useful for differentiating between species of rapidly growing mycobacteria, and for differentiating the genus Rhodococcus from the genus Nocardia. The majority of rhodococci tested were susceptible to 5-fluorouracil (20 micrograms ml-1), whereas the majority of nocardiae tested were resistant to it. Strains of Nocardia asteroides sensu stricto could be divided into two subgroups by their reaction to 5-fluorouracil.

Fluorouracil↗

PubMed1983

Serogrouping of Rhodococcus equi.

The serological relationships among 27 isolates of Rhodococcus equi selected from a total of 1,195 isolates were investigated by cross-agglutination and absorption tests. The presence of capsular material was demonstrated in all the 27 isolates by electron microscopic observation. Antisera were prepared by employing formalized antigen of each isolate. In the cross-agglutination test with formalized antigen, 13 antisera reacted with homologous antigens alone, but the remaining 14 antisera reacted not only with homologous antigens but also with one to four heterologous antigens. When these 14 antisera possessing heterologous agglutinins were absorbed with each of the cross-reacting antigens, 14 specific antisera were obtained. The cross-agglutination test with these 27 antisera proved the 27 strains examined to be serologically distinct from one another. These strains were designated serogroups 1 to 27. Thus the same number of grouping antisera were prepared. The distribution of each serogroup among the 1,195 isolates and 15 reference strains was investigated by the slide agglutination test. All the strains were found to be groupable. Most of them belonged to serogroups 1 to 4, 7 to 9, 11, 14, and 15. Of the serogroups designated, 4, 16, 2, 12, 21, 1, and 9 were identical with Prescott's serovars 1, 2, 3, 4, 5, 6, and 7, respectively.

Agglutination Tests↗

PubMed1985

Correlation of in vitro properties of Rhodococcus (Corynebacterium) equi with virulence for mice.

To study the virulence of Rhodococcus (Corynebacterium) equi, seven ATCC strains of different serotypes were tested for their LD50 in mice, clearance of the organism from the lungs and spleen following intravenous or intratracheal inoculation, and in vitro interaction with murine peritoneal macrophages. Strains ATCC 33704 and 33705 were virulent for mice and multiplied in the lungs and spleen, resulting in death of the animal in 5 days. The other five strains were avirulent for mice. The number of bacteria in the lungs and spleen of mice given these five strains decreased immediately. Pulmonary clearance of strains ATCC 33703, 33706, and 33707 was significantly more rapid than that of the virulent strains ATCC 33704 and 33705 12 hr after inoculation. Complete clearance of the avirulent strain ATCC 33707 occurred by day 14, while that of virulent ATCC 33704 and 33705 strains occurred by day 30. The virulent strains ATCC 33704 and 33705 were resistant not only to phagocytosis but also to intracellular killing by macrophages. Strains ATCC 33702 and 33706 were rapidly killed by macrophages although they were rather resistant to phagocytosis. Strain ATCC 33703 was easily phagocytized though resistant to killing by macrophages. The most avirulent strains, ATCC 33707 and 6939, were easily phagocytized and rapidly killed by macrophages. These results indicate that virulence appeared to be related to the ability of the organisms to resist clearance from the lungs and spleen and to resist phagocytosis and intracellular killing by macrophages.

Animals↗

PubMed1986

The metabolism of carbaryl by three bacterial isolates, Pseudomonas spp. (NCIB 12042 & 12043) and Rhodococcus sp. (NCIB 12038) from garden soil.

At an alkaline pH and in aqueous solution, carbaryl hydrolyses to form 1-naphthol, methylamine and carbon dioxide, but it is much more stable at an acid pH. Two bacterial isolated from garden soil, Pseudomonas sp. (NCIB 12042) and Rhodococcus sp. (NCIB 12038), could grow on carbaryl as sole carbon and nitrogen source at pH 6.8 but failed to metabolize carbaryl rapidly. Both could use 1-naphthol as sole carbon source and NCIB 12042 metabolized 1-naphthol via salicylic acid which induced higher expression of enzymes in the pathway. Strain NCIB 12038 metabolized 1-naphthol via salicylic and gentisic acids. In contrast, Pseudomonas sp. (NCIB 12043) was selected in a soil perfusion column enrichment at pH 5.2 and metabolized carbaryl rapidly to 1-naphthol and methylamine. 1-Naphthol was metabolized via gentisic acid. Neither salicylate nor gentisate induced higher expression of enzymes for 1-naphthol catabolism in NCIB 12038 and NCIB 12043.

Carbaryl↗

PubMed1985

The formaldehyde dehydrogenase of Rhodococcus erythropolis, a trimeric enzyme requiring a cofactor and active with alcohols.

During growth on compounds containing methyl groups a formaldehyde dehydrogenase is induced in the gram-positive bacteria Rhodococcus erythropolis. This formaldehyde dehydrogenase has been purified to homogeneity using affinity chromatography and permeation chromatography. The isoelectric point of the enzyme was 4.7. The molar mass of the native enzyme was determined as 130 000 g/mol. Sodium dodecyl sulfate gel electrophoresis yielded a single subunit with a molar mass of 44000 g/mol. These results, together with cross-linking experiments which yielded monomer, dimer, and trimer bands, are consistent with a trimeric subunit structure of the formaldehyde dehydrogenase. A heat-stable cofactor of low molar mass was required for activity with formaldehyde as substrate. This cofactor was found to be oxidizable, but active only in its reduced form. Preparative electrofocusing revealed that the cofactor is a weak acid with a pK of about 6.5. The enzyme was active with the homologous series of the primary alcohols, ethanol up to octanol, without requiring the presence of the cofactor. A mutant without formaldehyde dehydrogenase activity was not impaired in its growth with ethanol as substrate. It is suggested that the alcohols mimic the true substrate of the formaldehyde dehydrogenase, which could be a hydroxymethyl derivative of the cofactor, resulting from the addition of formaldehyde.

Actinomycetales↗

PubMed1995

Rhodococcus equi Virulence-Associated Antigens and Specific Antibody Response in AIDS Patients Infected with R. equi.

OBJECTIVES: To analyze the expression of the 15- to 17-kDa plasmid-encoded antigens from Rhodococcus equi isolates of 7 AIDS patients and determine the immunologic response to these proteins in the patients' sera. METHODS: The expression of the virulence proteins in R. equi isolates and the specific antibody response were investigated by immunoblotting. Plasmid DNA was analyzed by agarose gel electrophoresis. RESULTS: The only patient infected with a strain carrying the virulence 85-kb plasmid and expressing the 15- to 17-kDa antigens developed a fatal pneumonia and did not produce specific antibodies to the virulence proteins. Of the 6 patients infected with R. equi strains lacking both proteins and plasmid, only 1 subject had a pulmonary disease with poor clinical outcome and exhibited a negligible humoral immune response to R. equi antigenic components, whereas the other patients who produced a remarkable antibody response developed either an asymptomatic infection (1 case) or pneumonia (4 cases) which completely cleared up. CONCLUSIONS: Our findings suggest that R. equi disease can be induced without the expression of the 15- to 17-kDa virulence-associated plasmid-encoded antigens in HIV-infected patients with very low CD4+ cell counts. Nevertheless, both the synthesis of the virulence proteins and a defective humoral immune response to R. equi may contribute to the severity of rhodococcal disease.

Journal Article↗

PubMed1997

Rhodococcus equi infection in non-HIV-infected patients. Two case reports and review.

OBJECTIVE: To review two recent cases in HIV-negative subjects in the light of literature reports (52 patients without HIV infection till 1994). METHODS: Epidemiology (animal contacts, risk factors, year, country), clinical presentation, diagnostic methods (X-ray, tomography, microbiological techniques), therapeutic approach (antibiotics, surgery) and outcome were evaluated on the basis of clinical literature reports. RESULTS: Tumors constituted an important predisposing factor and less frequently hepatobiliary pathology, rheumatologic diseases, iatrogenic causes, psychiatric pathology and trauma. Exposure to animals was reported by 55% of the patients. Pneumonia and pleurisy, without preferential localization, were detected in 50% of the patients. Etiologic diagnosis was usually obtained after an invasive collection. Combined medical therapy and surgery were required by 27.8% of the patients, and 16.7% of the patients died. CONCLUSIONS: In recent years the number of Rhodococcus equi cases has been rising also in HIV-negative patients. The infection is ubiquitous. Accurate diagnosis and the prompt selection of the most appropriate therapy depend on close cooperation between clinicians and microbiologists.

Journal Article↗

PubMed1982

Capsular serotypes of Rhodococcus equi.

One hundred strains of Rhodococcus equi from various animal species and sources in Australia were examined for capsular serotype. Eighty-four of the strains fell into the existing 7 serotypes, and just under half of the strains belonged to serotype 1. Isolates from the intestines and faeces of horses, cattle, pigs and other species, and from soil, were found to belong to the same serotypes as those recovered from the lungs of foals with R. equi pneumonia. There was no clear relationships between capsular serotype and source of origin of the isolates.

Actinomycetales↗

PubMed1982

Chemical and Physical Characterization of Interfacial-Active Lipids from Rhodococcus erythropolis Grown on n-Alkanes.

Lipophilic compounds of the culture suspension containing Rhodococcus erythropolis DSM43215 had surfactant properties when the bacteria were cultivated with n-alkanes as the sole carbon source. Thirteen main components from a dichloromethane-methanol extract of the R. erythropolis cultures were isolated and characterized to specify quantitatively their surfactant properties, e.g., minimum surface and interfacial tensions and critical micelle concentrations. The interfacial activity of the organic extract was dominated by alpha,alpha-trehalose-6,6'-dicorynomycolates which reduced interfacial tension from 44 to 18 mN/m. Phosphatidylethanolamines which were also present in the organic extract reduced the interfacial tension below 1 mN/m. The trehalose corynomycolates had extremely low critical micelle concentrations in high-salinity solutions, and the interfacial properties were stabile in solutions with a wide range of pH and ionic strength.

Journal Article↗

PubMed1983

Investigation of the survival characteristics of Rhodococcus coprophilus and certain fecal indicator bacteria.

Rhodococcus coprophilus and Clostridium perfringens survived in fresh water samples held at 5, 20, and 30 degrees C for over 17 weeks, whereas Escherichia coli and fecal streptococci disappeared after 5 weeks at all three temperatures. R. coprophilus survived for more than 8 months in sterilized sewage and deionized water at all three temperatures, whereas in normal sewage held at 20 degrees C, the survival time was 12 to 26 weeks. In samples held at 30 degrees C, survival times were shorter, probably because of interbacterial competition or protozoal predation. The results indicate that R. coprophilus may be a useful indicator of the presence of remote fecal pollution of farm animal origin, but not of recent pollution, when enumerated alone in polluted waters or wastewaters.

Actinomycetales↗