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PubMed2006

The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse.

Rhodococcus sp. RHA1 (RHA1) is a potent polychlorinated biphenyl-degrading soil actinomycete that catabolizes a wide range of compounds and represents a genus of considerable industrial interest. RHA1 has one of the largest bacterial genomes sequenced to date, comprising 9,702,737 bp (67% G+C) arranged in a linear chromosome and three linear plasmids. A targeted insertion methodology was developed to determine the telomeric sequences. RHA1's 9,145 predicted protein-encoding genes are exceptionally rich in oxygenases (203) and ligases (192). Many of the oxygenases occur in the numerous pathways predicted to degrade aromatic compounds (30) or steroids (4). RHA1 also contains 24 nonribosomal peptide synthase genes, six of which exceed 25 kbp, and seven polyketide synthase genes, providing evidence that rhodococci harbor an extensive secondary metabolism. Among sequenced genomes, RHA1 is most similar to those of nocardial and mycobacterial strains. The genome contains few recent gene duplications. Moreover, three different analyses indicate that RHA1 has acquired fewer genes by recent horizontal transfer than most bacteria characterized to date and far fewer than Burkholderia xenovorans LB400, whose genome size and catabolic versatility rival those of RHA1. RHA1 and LB400 thus appear to demonstrate that ecologically similar bacteria can evolve large genomes by different means. Overall, RHA1 appears to have evolved to simultaneously catabolize a diverse range of plant-derived compounds in an O(2)-rich environment. In addition to establishing RHA1 as an important model for studying actinomycete physiology, this study provides critical insights that facilitate the exploitation of these industrially important microorganisms.

Bacterial Proteins↗

PubMed1997

Molecular cloning, expression, and sequence analysis of the endoglycoceramidase II gene from Rhodococcus species strain M-777.

Endoglycoceramidase (EGCase (EC 3.2.1.123)) is a hydrolase that hydrolyzes the linkage between the oligosaccharide and ceramide of various glycosphingolipids. This paper describes the molecular cloning and expression of EGCase II, one of the isoforms of EGCases. The gene encoding EGCase II was obtained by screening of a genomic DNA library from Rhodococcus sp. strain M-777 constructed in pUC19 with oligonucleotide probes deduced from a partial amino acid sequence of the enzyme protein. Recombinant Escherichia coli cells in which the EGCase II gene was expressed produced 14 units of the enzyme per liter of culture medium but did not produce sphingomyelinase. Recombinant EGCase II was a functioning enzyme with substrate specificity identical to that of the wild-type enzyme. Sequence analysis showed the presence of an open reading frame of 1470 base pairs encoding 490 amino acids. The N-terminal region of the deduced amino acid sequence had the general pattern of signal peptides of secreted prokaryotic proteins. Interestingly, the consensus sequence in the active site region of the endo-1,4-beta-glucanase family A was found in the amino acid sequence of EGCase II.

Amino Acid Sequence↗

PubMed1999

Stereoselective carveol dehydrogenase from Rhodococcus erythropolis DCL14. A novel nicotinoprotein belonging to the short chain dehydrogenase/reductase superfamily.

A novel nicotinoprotein, catalyzing the dichlorophenolindophenol-dependent oxidation of carveol to carvone, was purified to homogeneity from Rhodococcus erythropolis DCL14. The enzyme is specifically induced after growth on limonene and carveol. Dichlorophenolindophenol-dependent carveol dehydrogenase (CDH) is a homotetramer of 120 kDa with each subunit containing a tightly bound NAD(H) molecule. The enzyme is optimally active at pH 5.5 and 50 degrees C and displays a broad substrate specificity with a preference for substituted cyclohexanols. When incubated with a diastereomeric mixture of (4R)- or (4S)-carveol, CDH stereoselectively catalyzes the conversion of the (6S)-carveol stereoisomers only. Kinetic studies with pure stereoisomers showed that this is due to large differences in V(max)/K(m) values and simultaneous product inhibition by (R)- or (S)-carvone. The R. erythropolis CDH gene (limC) was identified in an operon encoding the enzymes involved in limonene degradation. The CDH nucleotide sequence revealed an open reading frame of 831 base pairs encoding a 277-amino acid protein with a deduced mass of 29,531 Da. The CDH primary structure shares 10-30% sequence identity with members of the short chain dehydrogenase/reductase superfamily. Structure homology modeling with trihydroxynaphthalene reductase from Magnaporthe grisea suggests that CDH from R. erythropolis DCL14 is an alpha/beta one-domain protein with an extra loop insertion involved in NAD binding and a flexible C-terminal part involved in monoterpene binding.

Alcohol Oxidoreductases↗

PubMed2002

A novel lipoarabinomannan from the equine pathogen Rhodococcus equi. Structure and effect on macrophage cytokine production.

Rhodococcus equi is a major cause of foal morbidity and mortality. We have investigated the presence of lipoglycan in this organism as closely related bacteria, notably Mycobacterium tuberculosis, produce lipoarabinomannans (LAM) that may play multiple roles as virulence determinants. The lipoglycan was structurally characterized by gas chromatography-mass spectrometry following permethylation, capillary electrophoresis after chemical degradation, and (1)H and (31)P and two-dimensional heteronuclear nuclear magnetic resonance studies. Key structural features of the lipoglycan are a linear alpha-1,6-mannan with side chains containing one 2-linked alpha-d-Manp residue. This polysaccharidic backbone is linked to a phosphatidylinositol mannosyl anchor. In contrast to mycobacterial LAM, there are no extensive arabinan domains but single terminal alpha-d-Araf residue capping the 2-linked alpha-d-Manp. The lipoglycan binds concanavalin A and mannose-binding protein consistent with the presence of t-alpha-d-Manp residues. We studied the ability of the lipoglycans to induce cytokines from equine macrophages, in comparison to whole cells of R. equi. These data revealed patterns of cytokine mRNA induction that suggest that the lipoglycan is involved in much of the early macrophage cytokine response to R. equi infection. These studies identify a novel LAM variant that may contribute to the pathogenesis of disease caused by R. equi.

Animals↗

PubMed2004

Crystal structure of 4-chlorocatechol 1,2-dioxygenase from the chlorophenol-utilizing gram-positive Rhodococcus opacus 1CP.

The crystal structure of the 4-chlorocatechol 1,2-dioxygenase from the Gram-positive bacterium Rhodococcus opacus (erythropolis) 1CP, a Fe(III) ion-containing enzyme involved in the aerobic biodegradation of chloroaromatic compounds, has been solved by multiple wavelength anomalous dispersion using the weak anomalous signal of the two catalytic irons (1 Fe/257 amino acids) and refined at a 2.5 A resolution (R(free) 28.7%; R factor 21.4%). The analysis of the structure and its comparison with the structure of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus ADP1 (Ac 1,2-CTD) highlight significant differences between these enzymes. The general topology of the present enzyme comprises two catalytic domains (one for each subunit) related by a noncrystallographic 2-fold axis and separated by a common alpha-helical zipper motif consisting of five N-terminal helices from each subunit; furthermore the C-terminal tail is shortened significantly with respect to the known Ac 1,2-CTD. The presence of two phospholipids binding in a hydrophobic tunnel along the dimer axis is shown here to be a common feature for this class of enzyme. The active site cavity presents several dissimilarities with respect to the known catechol-cleaving enzyme. The catalytic nonheme iron(III) ion is bound to the side chains of Tyr-134, Tyr-169, His-194, and His-196, and a cocrystallized benzoate ion, bound to the metal center, reveals details on a novel mode of binding of bidentate inhibitors and a distinctive hydrogen bond network with the surrounding ligands. Among the amino acid residues expected to interact with substrates, several are different from the corresponding analogs of Ac 1,2-CTD: Asp-52, Ala-53, Gly-76, Phe-78, and Cys-224; in addition, regions of largely conserved amino acid residues in the catalytic cleft show different shapes resulting from several substantial backbone and side chain shifts. The present structure is the first of intradiol dioxygenases that specifically catalyze the cleavage of chlorocatechols, key intermediates in the aerobic catabolism of toxic chloroaromatics.

Amino Acid Sequence↗

PubMed2001

Isolation and characterisation of Rhodococcus erythropolis TA57 able to degrade the triazine amine product from hydrolysis of sulfonylurea pesticides in soils.

We isolated a bacterium capable of metabolising a methylated and methoxylated s-triazine ring as the only nitrogen source. On a weight basis, the s-triazine, commonly named triazine amine (TAM), constitutes approx. half of several sulfonylurea herbicides and is formed after hydrolysis of these herbicides. The isolate, strain TA57 was identified using multi-phasic taxonomy as a gram-positive Rhodococcus erythropolis. Strain TA57 mineralised over 50% 14C-labelled TAM within 4 days in growing cultures using all of the nitrogen for growth. The degradation capacity was found stable in cells grown on either tryptic soy broth agar plates or in minimal medium with NH4+. Among other s-triazines tested, only one other methylated, but de-methoxylated s-triazine amine supported growth. Inoculating 10(6) cells of TA57 per gram of soil (d.w.) resulted in 50% mineralisation of 14C labelled TAM (1 mg kg(-1)) within 25 days, in contrary to the indigenous population that mineralised only 6% in 50 days.

Biodegradation, Environmental↗

PubMed2003

Effects of culture conditions on the mycolic acid composition of isolates of Rhodococcus spp. from activated sludgefoams.

The influence of two different carbon sources and three incubation temperatures on the mycolic acid compositions of three Rhodococcus isolates from activated sludge was examined using Selective Ion Monitoring (SIM) gas chromatography-mass spectrometry (GC-MS). Considerable qualitative and quantitative differences were detected in the mycolic acid compositions of the three very closely related isolates grown under the same conditions. Culture age also affected both the chain lengths and proportions of saturated mycolic acids detected in cell extracts, but not in the same manner for each isolate. Mycolic acids generally were of shorter chain lengths in cells grown with Tween 80 compared to glucose grown cells in strain 11R but the opposite situation occurred with strains A7 and D5. In all three, the proportion of unsaturated mycolic acids decreased with increasing growth temperatures. The taxonomic relevance of these observations and possible explanations for the observed changes in mycolic acid composition under various culture conditions are discussed.

Bacterial Typing Techniques↗

PubMed2002

Rhodococcus equi brain abscess in an immunocompetent patient.

Rhodococcus equi brain abscesses usually occur in immunocompromised patients with prolonged and refractory pulmonary infections. Herein we report a case of R. equi brain abscess in a 67-y-old man without immunodepression. Our patient recovered after neurosurgical resection and prolonged antimicrobial therapy with vancomycin and trimethoprim-sulfamethoxazole.

Actinomycetales Infections↗

PubMed2004

Nucleotide sequence of a portion of the camphor-degrading gene cluster from Rhodococcus sp. NCIMB 9784.

Rhodococcus sp. NCIMB 9784 is a camphor-degrading Gram-positive organism originally isolated from activated sewage sludge. A 5.4kbp portion of a proposed camphor degradation gene cluster from this organism was cloned and its nucleotide sequence determined. Four open reading frames (ORFs) were identified encoding proteins possibly involved in camphor metabolism; sequence alignment of the translation products suggested that the ORFs encode for a ferredoxin reductase, acyl-CoA ligase, epimerase and an acyl-CoA dehydrogenase. The last three activities are thought to be involved in the poorly understood late stage of camphor degradation. Our findings are entirely consistent with the proposed formation of a branched 9-carbon acid intermediate (3,4,4-trimethyl-5-oxo-trans-2-hexenoic acid) which has been isolated from the fermentation broth of camphor-grown cells.

Amino Acid Sequence↗

PubMed2003

The Rhodococcus sp. cocaine esterase: a bacterial candidate for novel pharmacokinetic-based therapies for cocaine abuse.

Cocaine is a powerful central nervous stimulant and among the most abused of drugs. Despite decades of efforts, however, no effective pharmacological treatments are available against cocaine addiction or toxic effects. Classical receptor-antagonist therapeutic approaches have not yielded significant effects, although cocaine targets are well known, thus fostering development of alternative therapeutic strategies. Recent evidence indicates that a sensible approach for treatment of cocaine abuse could be to interfere with cocaine pharmacokinetics, i.e. by preventing the drug from reaching the receptors responsible for its biological effects. Administration of cocaine binding antibodies as well as catalytic antibodies and enzymes that hydrolyze cocaine represent potential alternative therapeutic approach(es). The discovery of the cocaine esterase from the strain MBI of the bacterium Rhodococcus sp. (cocE) could be a major breakthrough in this field; cocE hydrolyzes cocaine faster than any known cocaine esterase and catalytic antibody.

Carboxylesterase↗

PubMed2001

Rhodococcus equi infections in immunocompetent hosts: case report and review.

Rhodococcus equi is an unusual cause of infection in humans. Infection in immunocompetent patients is extremely rare-only 19 cases in immunocompetent hosts have been reported. Localized infections represent nearly 50% of reported cases. Pulmonary infections account for only 42% of infections in immunocompetent hosts, compared with 84% of infections in immunocompromised hosts. The mortality rate among immunocompetent patients is approximately 11%, compared with rates of 50%-55% among human immunodeficiency virus (HIV)-infected patients and 20%-25% among non-HIV-infected immunocompromised patients. Treatment of infections in immunocompetent hosts depends on the site of infection. Serious infections need to be treated with combinations of parenteral antibiotics, followed by combinations of oral antibiotics. Surgical treatment is necessary for certain types of local infections. We report a pulmonary infection due to R. equi in an immunocompetent patient, and we review all reported cases of R. equi infection in immunocompetent hosts.

Actinomycetales Infections↗

PubMed2002

Rhodococcus equi: an emerging pathogen.

More than 100 cases of Rhodococcus equi infection have been reported since the first description of human disease caused by this organism. The vast majority of patients infected with R. equi are immunocompromised, and two-thirds have human immunodeficiency virus infection. The clinical manifestations of R. equi infection are diverse, although 80% of patients have some pulmonary involvement. The organism is easily cultured from specimens of infected tissue or body fluid, but it may be misdiagnosed as a contaminant. Treatment is often prolonged, and relapses at distant sites are common. This article summarizes the history, diagnosis, clinical features, and treatment of infection with this emerging pathogen.

Actinomycetales Infections↗

PubMed2003

Molecular epidemiology of Rhodococcus equi of intermediate virulence isolated from patients with and without acquired immune deficiency syndrome in Chiang Mai, Thailand.

We investigated the prevalence of virulent Rhodococcus equi in clinical isolates from 69 sporadic cases (60 men, 8 women, and 1 patient of unknown sex) in Chiang Mai, Thailand, between 1993 and 2001. Fifty were human immunodeficiency virus (HIV) positive, 3 were HIV negative, and HIV status was unknown for 16. Fifty-two (75%) of 69 isolates were strains of intermediate virulence that contained the virulence-associated 20-kDa antigen, and 17 isolates (25%) were avirulent. No virulent strains with the virulence-associated 15-17-kDa antigens were identified. R. equi was isolated from HIV-positive patients' houses and those of their neighbors: avirulent strains were widespread, but only 1 strain of intermediate virulence was isolated. R. equi strains of intermediate virulence were isolated from 4 (0.8%) of 500 submaxillary lymph nodes from apparently healthy pigs in Chiang Mai. The routes of R. equi acquisition should be investigated from the viewpoint of zoonosis and public health.

Acquired Immunodeficiency Syndrome↗

PubMed1998

Vertebral osteomyelitis due to Rhodococcus equi in a liver transplant recipient.

Rhodococcus equi is a rare but well-documented cause of cavitary pneumonia in immunocompromised patients. In this report the first case of R. equi infection manifesting as vertebral osteomyelitis is described. A 39-year-old liver transplant recipient presented with recurrent pneumonia and a pleura-based lung abscess and subsequently developed osteomyelitis of the lower thoracic spine. Surgical debridement and prolonged treatment with rifabutin and clarithromycin resulted in clinical cure. In the literature, 12 other cases of R. equi infection in solid-organ transplant recipients have been reported. Ten of these patients had documented pulmonary disease and seven had extrapulmonary manifestations. Prolonged antibiotic therapy and surgical drainage resulted in clinical improvement in > 90% of the reported cases.

Actinomycetales Infections↗

PubMed1998

Report of invasive Rhodococcus equi infections in Taiwan, with an emphasis on the emergence of multidrug-resistant strains.

From November 1995 to October 1997, seven patients with invasive infections due to Rhodococcus equi were treated in Taiwan. Four patients had pulmonary lesions, and one each of the remaining three patients had a recurrent Port-A-Cath (Kabi-Pharmacia, North Ryde, New South Wales, Australia)-related bacteremia, a primary bacteremia, and a brain abscess. Three patients had underlying hematologic malignancies, and one each of the remaining four patients had diabetes mellitus, Waldenström's macroglobulinemia, long-term use of steroids, and AIDS. The 13 isolates of R. equi recovered from these patients were identified by using API Coryne System (bioMérieux, Marcy l'Etoile, France), VITEK GPI card (bioMérieux Vitek, Hazelwood, MO), supplemental biochemical tests, and cellular fatty acid chromatograms. Susceptibilities of these isolates to 16 antimicrobial agents, with use of the agar dilution method, varied; among them, amikacin and trimethoprim-sulfamethoxazole were the most active agents. Different random amplified polymorphic DNA (RAPD) patterns of isolates from different patients documented the lack of epidemiological relatedness of the causative organisms of these infections. This study confirms the emergence of multidrug-resistant R. equi infection in Taiwan and documents the relapsing or reactivating nature of this infection.

Actinomycetales Infections↗

PubMed1994

Pulmonary malacoplakia associated with Rhodococcus equi infection in a patient with acquired immunodeficiency syndrome.

Pulmonary malacoplakia associated with Rhodococcus equi, an opportunistic Gram-positive coccobacillus, is unusual. Although this patient is the ninth reported to have pulmonary malacoplakia, he is only the third patient with the acquired immunodeficiency syndrome who has been reported to have pulmonary malacoplakia associated with R equi infection. The patient was a 49-year-old man infected with the human immunodeficiency virus who on initial examination was found to have cavitary pneumonia in the right upper lobe of the lung. Histologic examination of an open-lung biopsy specimen revealed sheets of foamy intraalveolar macrophages that contained coccobacillary organisms and some "targetlike" cytoplasmic inclusions. Electron-microscopic studies showed Michaelis-Gutmann bodies, some of which contained residual bacterial fragments. Cultures of the lung tissue, urine, and blood yielded growth of Gram-positive coccobacilli subsequently identified as R equi. In immunocompromised patients, the possibility of malacoplakia should be considered in the differential diagnosis of diffuse pulmonary infiltrate composed of foamy intraalveolar macrophages.

Acquired Immunodeficiency Syndrome↗

PubMed1995

Rhodococcus equi--an increasingly recognized opportunistic pathogen. Report of 12 cases and review of 65 cases in the literature.

Rhodococcus equi, a gram-positive, weakly acid-fast coccobacillus, initially isolated from horses, is becoming increasingly recognized as an important pathogen for immunosuppressed human hosts since the first human case was reported in 1967. A review of the English medical literature yielded 53 cases. During the last 11 years, the microbiology laboratories of the authors isolated the organism from 12 patients. Of the total 65 cases, 60 occurred in immunosuppressed patients with HIV infection, malignant neoplasms, or chronic immunosuppressive therapy. The lung is the most common primary site of infection. Typically, the lesion is densely infiltrated by histiocytes with multiple microabscesses. Intracellular gram-positive coccobacilli are easily demonstrated. R equi grows well on routine non-selective media at 35 degrees C. Previously, many cases may have been missed because the organism resembles oropharyngeal commensal diphtheroids. Clinical information with gram and Kinyoun strains on fresh isolates is helpful in recognizing the possibility of R equi infection.

Acquired Immunodeficiency Syndrome↗

PubMedDate not supplied

Rhodococcus equi infection in patients with and without human immunodeficiency virus infection.

Rhodococcus equi is an uncommon pathogen in humans that has occasionally been reported to cause infection in individuals with impaired cellular immunity. We summarize 30 previously published reports of human infection with R. equi and describe one additional case in a patient with AIDS. Eleven (35%) of the patients discussed in this report had AIDS or human immunodeficiency virus (HIV) infection, which is emerging as the leading cause of immunosuppression in cases of R. equi infection. Seventy-seven percent of all patients had pneumonia due to R. equi, and the infiltrate frequently cavitated. When HIV-infected patients were compared with those not infected with the virus, symptoms, age, and frequency of pneumonia were similar. Sputum and blood cultures were more likely to be positive in HIV-infected patients. Individuals with HIV infection also had a higher incidence of simultaneous secondary infections and higher mortality than non-HIV-infected patients (54.5% vs. 20%). The rate of survival for all patients was 75% when antibiotics were combined with surgical resection of infected tissue; in comparison, the survival rate among patients receiving antibiotics alone was 61.1%.

Acquired Immunodeficiency Syndrome↗