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PubMed2002

Survival and naphthalene-degrading activity of Rhodococcus sp. strain 1BN in soil microcosms.

AIMS: The survival and activity of Rhodococcus sp. strain 1BN, inoculated into naphthalene-contaminated sandy-loam soil microcosms, were studied using classical and molecular methods. METHODS AND RESULTS: The naphthalene-degrading activity of 1BN in microcosms was examined through viable counts, CO2 production and naphthalene consumption, while its survival after inoculation was monitored by detecting the contemporary presence of alkane and naphthalene degradative genes and by analysing the 16S rDNA specific restriction profile. The inoculation of 1BN did not significantly enhance naphthalene degradation in the naphthalene-contaminated native soil, where 1BN maintained its catabolic activity also when in the presence of indigenous microflora. Instead the rate of naphthalene degradation by the inoculated 1BN was greater in sterile naphthalene-contaminated soil. The level of 1BN was only slightly higher after inoculation regardless of whether indigenous naphthalene-degrading bacteria were present or not and 1BN remained viable even when the substrate was depleted. CONCLUSIONS: This study documents the colonization and growth of 1BN in a non-sterile, naphthalene-added, sandy-loam soil having an active indigenous naphthalene-degrading population. SIGNIFICANCE AND IMPACT OF THE STUDY: An active and well-established naphthalene-degrading bacterial population in the native soil did not hamper the survival of the introduced 1BN that, through its activity, enhanced the mineralization rate of naphthalene.

Cytochrome P-450 CYP4A↗
PubMed2002

Evidence that RDX biodegradation by Rhodococcus strain DN22 is plasmid-borne and involves a cytochrome p-450.

AIMS: To investigate the biodegradation of the explosive compound RDX in Rhodococcus strain DN22, a bacterium previously isolated for its ability to grow on RDX as sole nitrogen source. METHODS AND RESULTS: Analysis of the rates of RDX degradation and nitrite production indicated that 2 mol nitrite were produced per mole RDX degraded. Cells of strain DN22 had the highest activity against RDX during the exponential phase and low activity in the stationary phase. Nitrite production from RDX was inhibited by metyrapone, menadione, piperonyl butoxide, n-octylamine and carbon monoxide and inducible by pyrrolidine, pyridine and atrazine. Acridine orange treatment yielded RDX-minus derivatives of strain DN22 at a curing rate of 1.5% and all of the cured derivatives had lost a large plasmid. CONCLUSIONS: RDX biodegradation in strain DN22 appears to involve a plasmid-encoded cytochrome p-450 enzyme. SIGNIFICANCE AND IMPACT OF THE STUDY: Plasmid-borne RDX degradation genes could potentially be transferred between bacteria. Our research into RDX metabolism in strain DN22 will facilitate future applications of this bacterium for bioremediation.

Acridine Orange↗
PubMed2001

The att locus of Rhodococcus fascians strain D188 is essential for full virulence on tobacco through the production of an autoregulatory compound.

The ability of Rhodococcus fascians strain D188 to provoke leafy gall formation on a variety of plant species is correlated with the linear plasmid pFiD188, on which different pathogenicity loci were identified. The att locus affects the severity of symptom development on tobacco, whereas the fas locus is essential for virulence. To gain insight into the function of the att locus, sequence and expression analyses were performed. The att locus contains nine open reading frames homologous to arginine and beta-lactam biosynthetic genes. att gene expression is transcriptionally induced by leafy gall extracts, but not by extracts of uninfected plants, and depends on the attR gene that encodes a LysR-type transcriptional regulator. The att locus proves to be essential for the formation of inducing factors (IFs) that are present in gall extracts. Because the induction of the fas locus also requires the presence of IFs in gall extracts, the att locus is proposed to play an important role in regulating the expression of the virulence loci of R. fascians.

Bacterial Proteins↗
PubMed2002

Molecular and functional characterization of kshA and kshB, encoding two components of 3-ketosteroid 9alpha-hydroxylase, a class IA monooxygenase, in Rhodococcus erythropolis strain SQ1.

9 alpha-Hydroxylation of 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) is catalysed by 3-ketosteroid 9 alpha-hydroxylase (KSH), a key enzyme in microbial steroid catabolism. Very limited knowledge is presently available on the KSH enzyme. Here, we report for the first time the identification and molecular characterization of genes encoding KSH activity. The kshA and kshB genes, encoding KSH in Rhodococcus erythropolis strain SQ1, were cloned by functional complementation of mutant strains blocked in AD(D) 9 alpha-hydroxylation. Analysis of the deduced amino acid sequences of kshA and kshB showed that they contain domains typically conserved in class IA terminal oxygenases and class IA oxygenase reductases respectively. By definition, class IA oxygenases are made up of two components, thus classifying the KSH enzyme system in R. erythropolis strain SQ1 as a two-component class IA monooxygenase composed of KshA and KshB. Unmarked in frame gene deletion mutants of parent strain R. erythropolis SQ1, designated strains RG2 (kshA mutant) and RG4 (kshB mutant), were unable to grow on steroid substrates AD(D), whereas growth on 9 alpha-hydroxy-4-androstene-3,17-dione (9OHAD) was not affected. Incubation of these mutant strains with AD resulted in the accumulation of ADD (30-50% conversion), confirming the involvement of KshA and KshB in AD(D) 9 alpha-hydroxylation. Strain RG4 was also impaired in sterol degradation, suggesting a dual role for KshB in both sterol and steroid degradation.

Amino Acid Sequence↗
PubMed2003

Deletion of vapA encoding Virulence Associated Protein A attenuates the intracellular actinomycete Rhodococcus equi.

Virulent strains of the facultative intracellular bacterium Rhodococcus equi isolated from young horses (foals) with R. equi pneumonia, carry an 80-90 kb virulence plasmid and express a highly immunogenic 15-17 kDa protein of unknown function called VapA (Virulence Associated Protein A). Recent sequencing of the virulence plasmid identified a putative pathogenicity island encoding a novel family of seven Vap proteins including VapA. These proteins exhibit a significant sequence similarity to each other but have no homologues in other organisms. In this study, we describe the construction of an R. equi mutant lacking a 7.9 kb DNA region spanning five vap genes (vapA, -C, -D, -E and -F ). This vap locus mutant was attenuated for virulence in mice as it was unable to replicate in vivo and was rapidly cleared in comparison to the virulent wild-type strain. Complementation analysis of the vap locus mutant showed that expression of vapA alone could restore full virulence, whereas expression of vapC, -D and -E could not. We subsequently constructed an R. equi strain lacking only the vapA gene and found that it was attenuated for growth in vivo to the same degree as the vap locus mutant. Unlike wild-type R. equi which replicates intracellularly, both of the mutant strains exhibited a growth defect in macrophages although their attachment to the macrophages was unaffected. These studies provide the first proof of a role for vapA in the virulence of R. equi, and demonstrate that its presence is essential for intracellular growth in macrophages.

Actinomycetales Infections↗
PubMed1999

Heterologous expression of alkene monooxygenase from Rhodococcus rhodochrous B-276.

Alkene monooxygenase (AMO) from Rhodococcus rhodochrous (formerly Nocardia corallina) B-276 is a three-component enzyme system encoded by the four-gene operon amoABCD. AMO catalyses the stereoselective epoxygenation of aliphatic alkenes, yielding primarily R enantiomers. The presumed site of alkene oxygenation is a dinuclear iron centre similar to that in the soluble methane monooxygenases of methanotrophic bacteria, to which AMO exhibits a significant degree of amino acid sequence identity. The AMO complex was not expressed in Escherichia coli, at least partly because that host did not produce all of the AMO polypeptides. Expression of AMO was achieved in Streptomyces lividans by cloning the AMO genes into the thiostrepton-inducible expression plasmid pIJ6021. No background of AMO activity was detected in S. lividans cells without amoABCD and expression of AMO activity, at a level comparable to that from wild-type R. rhodochrous B-276, coincided with appearance of the AMO subunits. Recombinant AMO activity in cell-free extracts of S. lividans was stimulated by the addition of NADH and produced R-epoxypropane with comparable enantiomeric excess to AMO purified from the original organism. Although the whole AMO complex could not be expressed in E. coli, the functional coupling protein (AmoB) and reductase (AmoD) were expressed individually in E. coli as fusions with glutathione S-transferase. The expression systems described here now allow structure/function studies on AMO to be carried out by site-directed mutagenesis.

Base Sequence↗
PubMed1999

Nitrile hydratase involved in aldoxime metabolism from Rhodococcus sp. strain YH3-3 purification and characterization.

Nitrile hydratase responsible for aldoxime metabolism from the E-pyridine-3-aldoxime degrading bacterium, Rhodococcus sp. strain YH3-3 was purified and characterized. Addition of cobalt ion was necessary for the formation of enzyme. The enzyme activity was highly induced not only by nitriles and amides but also by several aldoxime compounds. The enzyme was purified approximately 108-fold with a 16% yield from the cell-free extract of the strain. The native enzyme had a Mr of approximately 130 000 and consisted of two subunits (alpha-subunit, 27 100; beta-subunit, 34 500). The enzyme contained approximately 2 mol cobalt per mol enzyme; it showed a maximum activity at 60 degrees C and at 40 degrees C under the rate assay and end-point assay conditions, respectively, and was stable over a wide range of pH (pH 2.5-11.0). The enzyme had a wide substrate specificity: it acted on aliphatic saturated and unsaturated as well as aromatic nitriles. The N-terminus of the beta-subunit showed good sequence similarities with those of other nitrile hydratases. Nitrile hydratase is part of the metabolic pathway for aldoximes in microorganisms.

Amino Acid Sequence↗
PubMed2000

Nitrilase of Rhodococcus rhodochrous J1. Conversion into the active form by subunit association.

Nitrilase-containing resting cells of Rhodococcus rhodochrous J1 converted acrylonitrile and benzonitrile to the corresponding acids, but the purified nitrilase hydrolyzed only benzonitrile, and not acrylonitrile. The activity of the purified enzyme towards acrylonitrile was recovered by preincubation with 10 mM benzonitrile, but not by preincubation with aliphatic nitriles such as acrylonitrile. It was shown by light-scattering experiments, that preincubation with benzonitrile led to the assembly of the inactive, purified and homodimeric 80-kDa enzyme to its active 410-kDa aggregate, which was proposed to be a decamer. Furthermore, the association concomitant with the activation was reached after dialysis of the enzyme against various salts and organic solvents, with the highest recovery reached at 10% saturated ammonium sulfate and 50% (v/v) glycerol, and by preincubation at increased temperatures or enzyme concentrations.

Acrylonitrile↗
PubMed2001

Rapid immunohistochemical detection of Rhodococcus equi in impression smears from affected foals on postmortem examination.

The first objective of this study was to develop an immunohistochemical procedure for rapid detection of Rhodococcus equi in impression smears from affected organs of foals on postmortem examination. The second aim was to demonstrate whether R. equi can be detected in smears of tracheal exudates collected from the same foals using an immunohistochemical method. Impression smears and cryostat and paraffin-embedded sections were made from the lungs and mediastinal lymph nodes of three foals (A, B and C) that had died of respiratory disease caused by R. equi, and also from the caudal mesenteric lymph node of foal A. Impression smears were made from the tracheal exudates of all foals. An affinity purified rabbit IgG was used for the immunohistochemical demonstration of R. equi. This antibody reacted with serotype 1 of R. equi in Ouchterlony's immunodiffusion and in the passive haemagglutination test, but not with other serotypes or with Streptococcus equi ssp. equi or Staphylococcus aureus, and failed to give an immunohistochemical reaction with Mycobacterium bovis or M. paratuberculosis. The immunohistochemical method proved to be of identical sensitivity to bacterial culture; moreover, from the lungs and mediastinal lymph nodes of one foal, R. equi could only be detected by this method. R. equi was demonstrated in smears of the tracheal exudates of all three foals. The results of this study indicate that the immunohistochemical method may be used for the rapid detection of R. equi in impression smears from the affected organs, especially abscesses, obtained postmortem, and possibly as a tool for diagnosing R. equi pneumonia in live foals by examining smears of tracheal aspirates.

Actinomycetales Infections↗
PubMed2002

Cholesterol oxidase and resistance of Rhodococcus equi to peroxidative stress in vitro in the presence of cholesterol.

Rhodococcus equi is a well-characterized bacterial pathogen which lyses cell membranes with the help of cholesterol oxidase (CO). Survival in macrophages is warranted by its ability to resist reactive radicals via catalase and superoxide dismutase (SOD). Therefore, CO production in the absence or presence of 0.1 % cholesterol and sensitivity to exogenous hydrogen peroxide (H2O2) and superoxide anion (SOA) were tested in seven strains of R. equi in vitro. When R. equi strains were grown on agar plates with cholesterol, the bacterial growth [colony-forming units (cfu)/plate] did not increase significantly in comparison with the growth on plates without cholesterol. The activity of CO increased, significantly for extracellular CO. In subsequent experiments, R. equi strains grown on cholesterol were stressed with H2O2 or SOA so that approximately 10 % of cfu/plate survived. During stress induced by SOA, membrane CO and SOD activity increased significantly. Catalase activity increased 2-fold with H2O2 and 3-fold with SOA exposure. These data suggest that the presence of cholesterol induces CO in bacteria grown on agar plates. Catalase, SOD and even membrane-bound CO respond to reactive oxygen species.

Actinomycetales Infections↗
PubMed2000

Detection of genes for alkane and naphthalene catabolism in Rhodococcus sp. strain 1BN.

Rhodococcus sp. 1BN was isolated from a contaminated site and showed various biodegradative capabilities. Besides naphthalene, strain 1BN degraded medium- (C6) and long-chain alkanes (C16-C28), benzene and toluene, alone or when the hydrocarbons were mixed in equal proportions. The nucleotide sequence of an alk polymerase chain reaction (PCR) fragment revealed a 59% nucleotide homology to the Pseudomonas oleovorans alkB gene. The nar fragments were highly homologous to genes coding for large and small subunits of cis-naphthalene 1,2-dioxygenase (narAa and narAb) and to cis-naphthalene dihydrodiol dehydrogenase (narB) from other rhodococci. The oxidation of indene to cis-(1S,2R)-1,2-dihydroxyindan by toluene-induced cells allows to hypothesize that strain 1BN also carries a toluene dioxygenase-like system.

Alkanes↗
PubMed2003

Rhodococcus equi pneumonia in a heart transplant recipient in Korea, with emphasis on microbial diagnosis.

Rhodococcus equi is an opportunistic pathogen that usually causes infection in immunocompromised hosts. A heart transplant recipient who had been treated with amphotericin B for pulmonary aspergillosis showed newly developed multiple nodules with a central necrotic area in the right lower lobes. Cultures of several blood samples and an aspirate of the lung nodule yielded a Gram-positive coccobacillary bacterium, which was initially reported as a Corynebacterium species, but was later identified as R. equi by API CORYNE (bioMerieux SA, Marcy l'Etoile, France) and by demonstrating the production of 'equi factor'. The identification was subsequently confirmed by an R. equi-specific polymerase chain reaction (PCR). The patient was successfully treated with ciprofloxacin and azithromycin for 14 weeks. This is the first documented case of R. equi infection in Korea. There is a possibility of underestimation of R. equi infections due to the misidentification of the organism as a contaminating diphtheroid. Because R. equi will not respond to the conventional empirical therapy, the microbiology laboratory should identify R. equi in a timely manner. R. equi-specific PCR will be a useful confirmatory test in human infection.

Actinomycetales Infections↗
PubMed1997

New method for RNA isolation from actinomycetes: application to the transcriptional analysis of the alcohol oxidoreductase gene thcE in Rhodococcus and Mycobacterium.

A new protocol for the isolation of RNA from Rhodococcus and other actinomycetes such as Mycobacterium and Amycolatopsis was developed. The method is based on rapid lysis of cells in a high-speed reciprocal shaker using small abrasive particles followed by spin column purification of the lysate. This quick procedure yields RNA preparations suitable for functional studies. This was shown for the thcE gene of R. erythropolis NI86/21, which encodes a N,N'-dimethyl-4-nitrosoaniline-dependent alcohol oxidoreductase. The thcE transcript was detected by Northern hybridization in R. erythropolis, R. fascians, Mycobacterium chlorophenolicum and Mycobacterium smegmatis. The transcriptional start point of the gene was determined by primer extension of the R. erythropolis mRNA.

Alcohol Oxidoreductases↗
PubMedDate not supplied

Rhodococcus equi.

Rhodococcus equi is an important cause of subacute or chronic abscessating bronchopneumonia of foals up to 3-5 months of age. It shares the lipid-rich cell wall envelope characteristic of the mycolata, including Mycobacterium tuberculosis, as well as the ability of pathogenic members of this group to survive within macrophages. The possession of a large virulence plasmid in isolates recovered from pneumonic foals is crucial for virulence. The plasmid contains an 27 kb pathogenicity island (PI) that encodes seven related virulence-associated proteins (Vaps), including the immunodominant surface-expressed protein, VapA. Only PI genes are differentially expressed when the organism is grown in macrophages in vitro. Ten of the PI genes, including six Vap genes, have signal sequences, suggesting that they are exported from the cell to interact with the macrophage. Different PI genes are regulated by temperature, pH, iron, oxidative stress and probably also by magnesium, all environmental changes encountered after environmental R. equi are inhaled in dust and are ingested into macrophages in the lung. The basis of pathogenicity of R. equi is its ability to multiply in and eventually to destroy alveolar macrophages. Infectivity is largely or exclusively limited to cells of the monocyte-macrophage lineage. Current evidence suggests that infection of foals with virulent R. equi results in some foals in subversion of cell-mediated immunity and development of an ineffective and sometimes lethal Th2-based immune response. Significant progress has been made recently in the development of R. equi-E. coli shuttle vectors, transformation and random and site specific mutagenesis procedures, all of which will be important in molecular dissection of the mechanisms by which R. equi subverts normal macrophage killing mechanisms and cell-mediated immunity.

Actinomycetales Infections↗
PubMed2002

Medical management of pneumonia caused by Rhodococcus equi in a renal transplant recipient.

Rhodococcus equi is an animal pathogen that occasionally causes opportunistic infections in immunocompromised patients. The most common clinical picture is one of necrotizing pneumonia with a tendency toward cavitation and the formation of abscesses. We report a case of pneumonia caused by R equi in a renal transplant patient. An excellent response was shown to antibiotic treatment. Symptoms regressed, and the progressive disappearance of the lesion was confirmed on follow-up computed tomography scans. Surgical intervention or other invasive procedures were not required. To our knowledge, 14 cases of infection by R equi in solid-organ transplant patients have been described to date. Nine were recipients of a renal allograft. Surgery was required in many of these patients, and all the renal transplant recipients required the use of invasive therapeutic techniques, such as pleural drainage. This is the first case of a renal transplant recipient in whom radiologic presentation was as a solid nodule without ensuing cavitation that resolved exclusively with antibiotic treatment.

Actinomycetales Infections↗
PubMedDate not supplied

Immunohistochemical detection of virulence-associated Rhodococcus equi antigens in pulmonary and intestinal lesions in horses.

Two horses with Rhodococcus equi infection were examined post mortem by an immunohistochemical method (peroxidase-antiperoxidase; PAP) with a monoclonal antibody (Mab 10G5) to the 15-17 kDa antigen of R. equi. One of the horses was also examined bacteriologically, R. equi being isolated in culture. Immunolabelling with this Mab was marked and widespread. On the other hand, the immunohistochemical reactivity of infected macrophages with a polyclonal antibody specific for lysozyme was slight. Thus, Mab 10G5 would appear to be a useful diagnostic reagent in R. equi infection, with or without cultural confirmation.

Actinomycetales Infections↗
PubMed2002

Role of surgery in Rhodococcus equi pulmonary infections.

Rhodococcus equi (R. equi) is a soil-dwelling bacterium that is increasingly associated with pulmonary infections in immunocompromised patients. While antibiotic therapy remains the cornerstone of treatment, surgery combined with antibiotics has an important role in select cases. We report two cases of pulmonary R. equi in immunocompromised patients who were treated with antibiotics, followed by surgical resection of the infected tissues, and then long-term antibiotics. Both patients had excellent outcomes. We advocate surgical resection of grossly infected pulmonary tissue as an adjunct to antimicrobial therapy for treatment of this uncommon, but potentially lethal pathogen.

Abscess↗