Rhodococci

Biochemistry, 1999 Dec 7, 38(49), 16105 - 14
Haloalkane dehalogenases: structure of a Rhodococcus enzyme; Newman J et al.; The hydrolytic haloalkane dehalogenases are promising bioremediation and biocatalytic agents . Two general classes of dehalogenases have been reported from Xanthobacter and Rhodococcus . While these enzymes share 30% amino acid sequence identity, they have significantly different substrate specificities and halide-binding properties . We report the 1.5 A resolution crystal structure of the Rhodococcus dehalogenase at pH 5.5, pH 7.0, and pH 5.5 in the presence of NaI . The Rhodococcus and Xanthobacter enzymes have significant structural homology in the alpha/beta hydrolase core, but differ considerably in the cap domain . Consistent with its broad specificity for primary, secondary, and cyclic haloalkanes, the Rhodococcus enzyme has a substantially larger active site cavity . Significantly, the Rhodococcus dehalogenase has a different catalytic triad topology than the Xanthobacter enzyme . In the Xanthobacter dehalogenase, the third carboxylate functionality in the triad is provided by D260, which is positioned on the loop between beta7 and the penultimate helix . The carboxylate functionality in the Rhodococcus catalytic triad is donated from E141 . A model of the enzyme cocrystallized with sodium iodide shows two iodide binding sites; one that defines the normal substrate and product-binding site and a second within the active site region . In the substrate and product complexes, the halogen binds to the Xanthobacter enzyme via hydrogen bonds with the N(eta)H of both W125 and W175 . The Rhodococcusenzyme does not have a tryptophan analogous to W175 . Instead, bound halide is stabilized with hydrogen bonds to the N(eta)H of W118 and to N(delta)H of N52 . It appears that when cocrystallized with NaI the Rhodococcus enzyme has a rare stable S-I covalent bond to S(gamma) of C187.

Appl Environ Microbiol, 1999 Dec, 65(12), 5636 - 8
Regiospecific internal desaturation of aliphatic compounds by a mutant Rhodococcus strain; Koike K et al.; A mutant Rhodococcus strain lacking the ability to utilize 1-chlorohexadecane was found to cis-desaturate aliphatic compounds, such as 1-chlorohexadecane, n-hexadecane, and heptadecanonitrile, yielding corresponding products with a double bond mainly at the ninth carbon from the terminal methyl groups . A new oxidative pathway involving the cis-desaturation step was suggested for alkane utilization by Rhodococcus spp.

J Appl Microbiol, 1999 Oct, 87(4), 472 - 80
Identification and environmental detection of Rhodococcus species by 16S rDNA-targeted PCR; Bell KS et al.; Bacteria of the genus Rhodococcus can degrade a wide range of organic pollutants and catalyse many useful biotransformations . There is a need for improved tests to identify Rhodococcus species . PCR-based methods for species identification offer advantages in terms of speed and accuracy over traditional methods and can allow direct detection of microbes in environmental samples., PCR tests, using primers targeted at species-specific sequences in the 16S rRNA gene, were successfully developed for R . globerulus, R . erythropolis, R . opacus and R . ruber . These tests gave positive results with all or most strains of target species but did not generally cross-react with other species . Cases of apparent cross-reaction were shown to be due to prior misclassification of strains of R . opacus as R . erythropolis and of strains of R . ruber as R . rhodochrous . A simple and rapid method for the extraction and purification of DNA from soil was developed and successfully applied to the PCR detection of indigenous R . erythropolis in an environmental sample . Cell lysis in the samples was achieved by lysozyme and sarkosyl treatment, aided by freeze-thaw cycles . Removal of humic compounds inhibitory to PCR was accomplished by CTAB treatment with solvent extraction and, if necessary, passage of extracts through Sepharose CL-6B in a spun-column format . Extracts prepared using a tris-EDTA buffer were much clearer than those prepared using a sodium phosphate buffer, indicating lower levels of humic compounds . A detection limit of 104 cfu g-1 of soil was achieved and the use of a secondary PCR allowed detection of 1 cfu g-1.

J Antibiot (Tokyo), 1999 Aug, 52(8), 710 - 20
Rhodopeptins, novel cyclic tetrapeptides with antifungal activities from Rhodococcus sp . III . Synthetic study of rhodopeptins; Chiba H et al.; Total syntheses of cyclo (-Gly-L-Lys-L-Val-(R)-3-aminododecanoyl-); LV9nA and its diastereomer cyclo (-Gly-L-Lys-L-Val-(S)-3-aminododecanoyl-); LV9nB, congeners of rhodopeptin B5 on beta-amino acid moiety, were achieved . The beta-amino acid moiety was prepared as a racemate by the thermal Michael addition of an amine to alpha,beta-unsaturated ester . The racemic beta-amino acids were converted to their L-Valylamide derivatives and the obtained diastereomers were separated . Coupling of both diastereomers, L-Val-beta-amino acids with Gly-L-Lys gave linear tetrapeptides, and tetrapeptides were cyclized by diphenylphosphoryl azide (DPPA) method between C-terminus of beta-amino acid and N-terminus of Gly to give cyclic tetrapeptides . The deprotected cyclic tetrapeptides, LV9nA and LV9nB, both exhibited almost the same antifungal activity as the naturally obtained rhodopeptins . Furthermore, comparison of the 1H NMR spectra of two congeners and rhodopeptin B5 suggested that the stereochemistry of beta-amino acid moiety in natural rhodopeptin B5 has (R)-configuration.

J Antibiot (Tokyo), 1999 Aug, 52(8), 700 - 9
Rhodopeptins, novel cyclic tetrapeptides with antifungal activities from Rhodococcus sp . II . Structure elucidation; Chiba H et al.; The structures of rhodopeptins, novel antifungal peptides, were determined on the basis of physico-chemical analyses of the intact molecules and their acid hydrolysates . The structures of rhodopeptins C1, C2, C3, C4 and B5 were determined to be cyclo (-Gly-L-Orn-L-Val-3-amino-10-methyldodecanoyl-), cyclo (-Gly-L-Orn-L-Ile-3-amino-10-methyldodecanoyl-), cyclo (-Gly-L-Orn-L-Val-3-amino-12-methyltridecanoyl-), cyclo (-Gly-L-Orn-L-Val-3-amino- 12-methyltetradecanoyl-) and cyclo (-Gly-L-Lys-L-Val-3-amino-13-methyltetradecanoyl-), respectively . They are novel cyclic tetrapeptides containing a lipophilic beta-amino acid.

Carbohydr Res, 1999 Aug 15, 320(3-4), 209 - 22
The structure of the specific capsular polysaccharide of Rhodococcus equi serotype 4; Severn WB et al.; The specific capsular polysaccharide produced by Rhodococcus equi serotype 4 was found to be a high-molecular-weight acidic polymer composed of D-glucose, D-mannose, pyruvic acid and a previously unidentified 5-amino-3,5-dideoxynonulosonic (rhodaminic) acid in the proportions 2:1:1:1 . Structural analysis, employing a combination of microanalytical methods, nuclear magnetic resonance spectroscopy, and mass spectrometric techniques, established that the polysaccharide consisted of linear repeating tetrasaccharide units having the sequence of residues shown below . In the native polysaccharide, the rhodaminic acid residues were present as their acetamido derivatives (RhoANAc) and carried 1-carboxyethylidene groups that bridged the O-7 and O-9 positions . Treatment of the capsular polysaccharide with dilute acetic acid and/or anhydrous hydrogen fluoride under hydrolytic/solvolytic conditions, resulted in the formation of four different oligosaccharide species . The 1H and 13C NMR resonances of these oligosaccharide fragments and of the native serotype 4 capsular polysaccharides were fully assigned by homo- and heteronuclear chemical shift correlation methods.

Biochem J, 1999 Dec 1, 344 Pt 2, 349 - 58
alpha5 subunit in Trypanosoma brucei proteasome can self-assemble to form a cylinder of four stacked heptamer rings; Yao Y et al.; The proteasomes have a central role in catalysing protein degradation among both prokaryotes and eukaryotes . The 20 S proteasome constitutes their catalytic core . In studying the structure of Trypanosoma brucei 20 S proteasomes, we isolated by two-dimensional (2D) gel electrophoresis a 27 kDa subunit protein with an estimated pI of 4.7 and subjected it to mass spectrometric analysis . A tryptic peptide sequence from the protein was found identical with that of the rat alpha5 subunit . With the use of antiserum against T . brucei 20 S proteasomes to screen a T . b . rhodesiense lambda expression cDNA library, we obtained a cDNA clone encoding a full-length protein of 246 amino acid residues with a calculated molecular mass of 27174 Da and a pI of 4.71 . It bears 50 . 0% and 46.3% sequence identity with rat and yeast proteasome subunit alpha5 respectively, and matches all the peptide sequences derived from MS of the 2D gel-purified protein . The protein is thus designated the alpha5 subunit of T . brucei 20 S proteasome (TbPSA5) . The recombinant protein, expressed in plasmid-transformed Escherichia coli, was found in a 27 kDa monomer form as well as polymerized forms with estimated molecular masses ranging from 190 to 800 kDa . Under the electron microscope, the most highly polymerized forms bear the appearance of cylinders of four-stacked heptamer rings with an estimated outer diameter of 14.5 nm and a length of 18 nm, which were immunoprecipitable by anti-(T . brucei 20 S proteasome) antiserum . In view of the documented self-assembly of the archaeon proteasome alpha subunit into double heptamer rings and the spontaneous assembly of the two alpha subunits from the 20 S proteasome of Rhodococcus erythropolis, the self-assembly of the T . brucei alpha subunit might reflect a common feature of proteasome biogenesis shared by prokaryotes and primitive eukaryotes such as the trypanosomes but apparently lost among the higher forms of eukaryote such as the yeast and the mammals.

DNA Seq, 1999, 10(1), 61 - 6
Sequence analysis of the oxidase/reductase genes upstream of the Rhodococcus erythropolis aldehyde dehydrogenase gene thcA reveals a gene organisation different from Mycobacterium tuberculosis; Nagy I et al.; The sequence of the DNA region upstream of the thiocarbamate-inducible aldehyde dehydrogenase gene thcA of Rhodococcus erythropolis NI86/21 was determined . Most of the predicted ORFs are related to various oxidases/reductases, including short-chain oxidases/reductases, GMC oxidoreductases, alpha-hydroxy acid oxidases (subfamily 1 flavin oxidases/dehydrogenases), and subfamily 2 flavin oxidases/dehydrogenases . One ORF is related to enzymes involved in biosynthesis of PQQ or molybdopterin cofactors . In addition, a putative member of the TetR family of regulatory proteins was identified . The substantial sequence divergence from functionally characterized enzymes precludes a reliable prediction about the probable function of these proteins at this stage . In Mycobacterium tuberculosis H37Rv, most of these ORFs have homologs that are also clustered in the genome, but some striking differences in gene organization were observed between Rhodococcus and Mycobacterium.

FEMS Microbiol Lett, 1999 Dec 1, 181(1), 73 - 82
Preferential oxidative dehalogenation upon conversion of 2-halophenols by Rhodococcus opacus 1G; Bondar VS et al.; The regiospecificity of hydroxylation of C2-halogenated phenols by Rhodococcus opacus 1G was investigated . Oxidative defluorination at the C2 position ortho with respect to the hydroxyl moiety was preferred over hydroxylation at the non-fluorinated C6 position for all 2-fluorophenol compounds studied . Initial hydroxylation of 2,3, 5-trichlorophenol resulted in the exclusive formation of 3, 5-dichlorocatechol . These results indicate that, in contrast to all other phenol ortho-hydroxylases studied so far, phenol hydroxylase from R . opacus 1G is capable of catalyzing preferential oxidative defluorination but also oxidative dechlorination.

P R Health Sci J, 1999 Sep, 18(3), 285 - 8
Otomastoiditis caused by Rhodococcus equi in a patient with AIDS; Kim SC et al.; Rhodococcus equi is a well-recognized pathogen in veterinary medicine and a rare but well-documented cause of cavitary pneumonia in immunocompromised patients . Most cases of Rhodococcus equi infections in these patients involve the lungs . Otomastoiditis due to Rhodococcus equi is rare, and disseminated Rhodococcus equi with otomastoiditis has never been reported . We report a case of otomastoiditis with systemic dissemination due to Rhodococcus equi in a patient with AIDS.

Appl Environ Microbiol, 1999 Nov, 65(11), 4967 - 72
Microbial desulfurization of alkylated dibenzothiophenes from a hydrodesulfurized middle distillate by Rhodococcus erythropolis I-19; Folsom BR et al.; Rhodococcus erythropolis I-19, containing multiple copies of key dsz genes, was used to desulfurize alkylated dibenzothiophenes (Cx-DBTs) found in a hydrodesulfurized middle-distillate petroleum (MD 1850) . Initial desulfurization rates of dibenzothiophene (DBT) and MD 1850 by I-19 were 5.0 and 2.5 micromol g dry cell weight(-1) min(-1), more than 25-fold higher than that for wild-type bacteria . According to sulfur K-edge X-ray absorption near-edge structure (XANES) analysis, thiophenic compounds accounted for >95% of the total sulfur found in MD 1850, predominantly Cx-DBTs and alkylated benzothiophenes . Extensive biodesulfurization resulted in a 67% reduction of total sulfur from 1,850 to 615 ppm S . XANES analysis of the 615-ppm material gave a sulfur distribution of 75% thiophenes, 11% sulfides, 2% sulfoxides, and 12% sulfones . I-19 preferentially desulfurized DBT and C1-DBTs, followed by the more highly alkylated Cx-DBTs . Shifting zero- to first-order (first-order) desulfurization rate kinetics were observed when MD 1850 was diluted with hexadecane . Apparent saturation rate constant (K(0)) and half-saturation rate constant (K(1)) values were calculated to be 2.8 micromol g dry cell weight(-1) min(-1) and 130 ppm, respectively . However, partial biocatalytic reduction of MD 1850 sulfur concentration followed by determination of initial rates with fresh biocatalyst led to a sigmoidal kinetic behavior . A competitive-substrate model suggested that the apparent K(1) values for each group of Cx-DBTs increased with increasing alkylation . Overall desulfurization rate kinetics with I-19 were affected by the concentration and distribution of Cx-DBTs according to the number and/or lengths of alkyl groups attached to the basic ring structure.

Acta Biol Hung, 1998, 49(2-4), 405 - 12
Relationships between demethylase activity, formaldehyde and oxygen during incubation of Rhodococcus erythropolis with veratrate; Pazdzioch-Czochra M et al.; Relationships between demethylase activity, formaldehyde and oxygen were investigated . Demethylase activity was measured against the following substrates: veratric, vanillic, and isovanillic acids, as well as in the presence of guaiacol . The influence of ATP and GTP on demethylase activity was also checked . Demethylase activity was found to be dependent on the capability of the cells for endogenous oxygen uptake . In some cases ATP produced the opposite effect: instead of being taken up, oxygen was released, which suggested a reversibility of the demethylation reaction . Curiously enough, GTP demonstrated the same effect . Changes in enzyme activity were correlated with those occurring in the level of formaldehyde . The latter increased after addition of ATP, but decreased after addition of GTP.

Neuroradiology, 1999 Sep, 41(9), 699 - 701
Severe otitis and mastoiditis due to Rhodococcus equi in a patient with AIDS . Case report; Ibarra R et al.; We report a case of otitis media associated with pneumonia due to Rhodococcus equi . A 31-year-old patient with AIDS presented with cough and right facial palsy . Imaging revealed right otitis media and severe temporal bone destruction, associated with pneumonia . R . equi was isolated from ear secretions, blood, and sputum . The radiologic findings are described . This unusual pathogen should be included in the differential diagnosis of the immunocompromised patient with aggressive otitis.

Microbiol Immunol, 1999, 43(8), 785 - 93
Production and partial characterization of antibody to cord factor (trehalose 6,6'-dimycolate) in mice; Fujiwara N et al.; Antibody production against the trehalose 6,6'-dimycolate (TDM, cord factor) of Rhodococcus ruber, a non-pathogenic species of the Actinomycetales group, was investigated in mice by repeated intraperitoneal injection of TDM in water-in-oil-in-water micelles without carrier protein . The antigenic TDM was isolated and purified chromatographically from the chloroform-methanol extractable lipids of R . ruber . The hydrophobic moiety of this TDM was composed of two molecules of monoenoic or dienoic alpha-mycolic acids with a carbon chain length ranging from C44 to C48 centering at C46 . To detect the antibody, an enzyme-linked immunosorbent assay (ELISA) system was employed using plastic plates coated with TDM . The antibody reacted against the TDM of R . ruber . The antibody was reactive in similar fashion against glycosyl monomycolates differing in the carbohydrate moiety, such as that of glucose mycolate (GM) and mannose mycolate (MM), obtained from R . ruber . Moreover, the antibody reacted against mycolic acid methyl ester itself when it was used as the antigen in ELISA, and trehalose did not absorb the antibody to TDM or inhibit the reaction . These results indicate that the epitope of TDM recognized by the antibody is mycolic acid, an extremely hydrophobic part of the molecule . Next, we prepared monoclonal anti-TDM antibody (moAb) in mice myeloma cells to examine its biological activities and the role of humoral immunity in mycobacterial infection . MoAb reacted against the TDM, glycosyl mycolate, and mycolic acid methyl ester in ELISA in the same manner as our polyclonal antibody did . The administration of moAb suppressed granuloma formation in the lungs, spleen, and liver induced by TDM and inhibited the production of interleukin-1 (IL-1) and chemotactic factor, which is reported to precede granuloma formation.

J Biochem (Tokyo), 1999 Oct, 126(4), 662 - 7
Essential tyrosine residues in 3-ketosteroid-delta(1)-dehydrogenase from Rhodococcus rhodochrous; Fujii C et al.; Tetranitromethane treatment of 3-ketosteroid-Delta(1)-dehydrogenase of Rhodococcus rhodochrous caused loss of the catalytic activity in a time- and concentration-dependent manner . Peptides (P-81) and (PN-83) were isolated from tryptic digests of the native and tetranitromethane-treated enzyme proteins, respectively . PN-83 was the nitrated form of P-81 . The amino acid sequence was GGAPLIDYLESDDDLEFMVYPWPDYFGK (positions 97-124 of the dehydrogenase sequence) . PN-83 showed a low yield of PTH-Tyr of position 116, i.e . less than 5% of that of P-81, and instead a high yield of PTH-3-nitrotyrosine . This indicated that tetranitromethane modifies Y-116 under the experimental conditions used . Mutation of Y-104, Y-116, and Y-121 to smaller amino acid residues, Phe, Ser, or Ala, significantly changed the catalytic activity of the dehydrogenase . All of the mutants contained FAD and exhibited the same spectrophotometric properties as those of the wild type enzyme . The K(m) values for 4-androstene-3,17-dione of the Y-104, Y-116, and Y-121 mutants changed to large values . The most drastic change was observed for Y116A . The K(d) values for 1,4-androstadiene-3,17-dione of the Y116 mutants changed to 1.5-2.6-fold larger values than that of the recombinant enzyme . The Y-121 mutant enzymes exhibited catalytic activities like those of the recombinant enzyme, but the catalytic efficiencies of Y121F and Y121A drastically decreased to 0 . 014-0.054% of that of the recombinant enzyme . The present results indicate that Y-121 plays an important role in the catalytic function, and that Y-116 and Y-104 act on binding of the substrate steroid.

Infect Immun, 1999 Oct, 67(10), 5041 - 7
Modulation of cytokine response of pneumonic foals by virulent Rhodococcus equi; Giguere S et al.; The ability of Rhodococcus equi to induce pneumonia in foals depends on the presence of an 85- to 90-kb plasmid . In this study, we evaluated whether plasmid-encoded products mediate virulence by modulating the cytokine response of foals . Foals infected intrabronchially with a virulence plasmid-containing strain of R . equi had similar gamma interferon (IFN-gamma) and interleukin-12 (IL-12) p35 but significantly higher IL-1beta, IL-10, IL-12 p40, and tumor necrosis factor alpha (TNF-alpha) mRNA expression in lung tissue compared to foals infected with the plasmid-cured derivative . IFN-gamma mRNA expression levels in CD4+ T lymphocytes isolated from bronchial lymph nodes (BLN) were similar for the two groups of R . equi-infected foals on day 3 postinfection . However, on day 14, in association with pneumonia and marked multiplication of virulent R . equi but with complete clearance of the plasmid-cured derivative, IFN-gamma mRNA expression in BLN CD4+ T lymphocytes was significantly (P < 0.001) higher in foals infected with the plasmid-cured derivative . These results suggests an immunomodulating role for R . equi virulence plasmid-encoded products in downregulating IFN-gamma mRNA expression by CD4+ T lymphocytes.

Steroids, 1999 Aug, 64(8), 535 - 40
Nitrile hydratase from Rhodococcus erythropolis: metabolization of steroidal compounds with a nitrile group; Kaufmann G et al.; The progestin dienogest (17alpha-cyanomethyl-17beta-hydroxy-estra-4,9-dien-3-one) was metabolized by the nitrile hydratase-containing microorganism Rhodococcus erythropolis . An enzymatic hydrolysis of the nitrile group at the 17alpha-side chain was intended to obtain novel derivatives and to test them for progesterone receptor affinity . In contrast to the rapid enzymatic hydrolysis of nonsteroidal nitriles, the nitrile group of dienogest was cleaved very slowly . The dominant reaction was an aromatization of ring A . After prolonged fermentation, the 17alpha-acetamido derivatives of estradiol and of 9(11)-dehydroestradiol were formed . Three of the metabolites were also prepared synthetically . They were tested for hormonal activity by assessing their binding to progesterone and estrogen receptors in vitro . Neither the aromatized 17alpha-acetamido derivatives nor the dienogest derivative 17alpha-acetamido-17beta-hydroxy-estra-4,9-dien-3-one, which was prepared synthetically only, exhibited affinity for the progesterone receptor.

Biochem Biophys Res Commun, 1999 Sep 24, 263(2), 460 - 4
Purification and partial characterization of caffeine oxidase--A novel enzyme from a mixed culture consortium; Madyastha KM et al.; Cell-free extract prepared from a mixed culture consisting of strains belonging to the genera Klebsiella and Rhodococcus grown in the presence of caffeine contains a novel enzyme, caffeine (1,3, 7-trimethylxanthine) oxidase which catalyzes the oxidation of caffeine at the C-8 position to produce 1,3,7-trimethyluric acid . The enzyme was purified to homogeneity by a combination of ion-exchange and hydrophobic column chromatographies . Both native and SDS/PAGE of the purified enzyme showed a single protein band and the subunit molecular mass of the protein was determined to be 85 kDa . Dichlorophenol indophenol and cytochrome c served as good electron acceptors but NAD and NADP did not . Caffeine served as the best substrate with an apparent K(m) of 11.4 microM . various analogues of theobromine were also effective substrates for caffeine oxidase . The activity was inhibited by o-phenanthroline, H(2)O(2), and methanol, but salicylate, thiol-group blocking reagents, and sodium arsenite, the known xanthine oxidase inhibitors, did not inhibit the reaction . The spectral characteristics of the purified enzyme suggest that it is a flavoprotein containing non-heme iron .

J Immunol, 1999 Oct 1, 163(7), 3883 - 9
TNF receptor p55 is required for elimination of inflammatory cells following control of intracellular pathogens; Kanaly ST et al.; The elimination of lymphocytes within inflammatory lesions is a critical component in the resolution of disease once pathogens have been cleared . We report here that signaling through the TNF receptor p55 (TNFRp55) is required to eliminate lymphocytes from lesions associated with intracellular pathogens . Thus, TNFRp55-/- mice, but not Fas-deficient mice, maintained inflammatory lesions associated with either Leishmania major or Rhodococcus equi infection, although they developed a Th1 response and controlled the pathogens . Inflammatory cells from either L . major- or R . equi-infected C57BL/6 mice were sensitive to TNF-induced apoptosis, and conversely the number of apoptotic cells in the lesions from TNFRp55-/- mice was dramatically reduced compared with wild-type mice . Furthermore, in vivo depletion of TNF in wild-type mice blocked lesion regression following R . equi infection . Taken together, our results suggest that signaling through the TNFRp55, but not Fas, is required to induce apoptosis of T cells within inflammatory lesions once pathogens are eliminated, and that in its absence lesions fail to regress.

J Clin Microbiol, 1999 Oct, 37(10), 3417 - 20
Restriction fragment length polymorphisms of virulence plasmids in Rhodococcus equi; Takai S et al.; Virulent Rhodococcus equi, which is a well-known cause of pyogranulomatous pneumonia in foals, possesses a large plasmid encoding virulence-associated 15- to 17-kDa antigens . Foal and soil isolates from five countries-Argentina, Australia, Canada, France, and Japan-were investigated for the presence of 15- to 17-kDa antigens by colony blotting, using the monoclonal antibody 10G5, and the gene coding for 15- to 17-kDa antigens by PCR . Plasmid DNAs extracted from positive isolates were digested with restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII, and the digestion patterns that resulted divided the plasmids of virulent isolates into five closely related types . Three of the five types had already been reported in Canadian and Japanese isolates, and the two new types had been found in French and Japanese isolates . Therefore, we tentatively designated these five types 85-kb type I (pREAT701), 85-kb type II (a new type), 87-kb type I (EcoRI and BamHI type 2 {V . M . Nicholson and J . F . Prescott, J . Clin . Microbiol . 35:738-740, 1997}), 87-kb type II (a new type), and 90-kb (pREL1) plasmids . The 85-kb type I plasmid was found in isolates from Argentina, Australia, Canada, and France . Plasmid 87-kb type I was isolated in specimens from Argentina, Canada, and France . The 85-kb type II plasmid appeared in isolates from France . On the other hand, plasmids 87-kb type II and 90-kb were found only in isolates from Japan . These results revealed geographic differences in the distribution of the virulence plasmids found in the five countries and suggested that the restriction fragment length polymorphism of virulence plasmids might be useful to elucidate the molecular epidemiology of virulent R . equi in the world.

Rev Pneumol Clin, 1999 Jun, 55(3), 171 - 4
{Pulmonary malacoplakia caused by Rhodococcus equi in AIDS: a case report}; Goupil F et al.; We describe the observation of a right upper lobe consolidation with cavitation produced by Rhodococcus equi in a patient suffering from AIDS . The inefficacy of a prolonged antimicrobial therapy adapted against R . equi led to a right upper lobectomy . The histopathology showed a pseudotumoral mass, with dense infiltration of macrophages containing Michaelis-Gutmann bodies, which was positive for the culture of R . equi . Pulmonary malacoplakia with Rhodococcus equi was diagnosed . This pathology should be evoked when a R . equi pneumonia persists despite a right management of treatment for several months . The features of pneumonia with Rhodococcus equi and of pulmonary malacoplakia are taken from a literature review.

Med Vet Entomol, 1999 May, 13(2), 115 - 9
Expression of a functional antibody fragment in the gut of Rhodnius prolixus via transgenic bacterial symbiont Rhodococcus rhodnii; Durvasula RV et al.; Expression within insects of foreign antiparasitic gene products via microbial symbionts could be used to prevent transmission of vector-borne pathogens to vertebrate hosts . Genetically transformed symbiotic bacteria Rhodococcus rhodnii expressed functional antibody fragments (rDB3 encoding murine V(H)/K which binds progesterone) that were exported into the gut lumen of the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae), a vector of Chagas disease . Transgenic symbionts were maintained in successive nymphal instars and adults of Rhodnius prolixus despite competition with native untransformed Rhodococcus rhodnii . This is the first description of a functional mammalian antibody fragment expressed in an insect . Our system is a model for constructing paratransgenic insects (insects carrying transformed symbionts) with compromised ability to transmit pathogens.

J Biol Chem, 1999 Sep 10, 274(37), 26296 - 304
Stereoselective carveol dehydrogenase from Rhodococcus erythropolis DCL14 . A novel nicotinoprotein belonging to the short chain dehydrogenase/reductase superfamily; van der Werf MJ et al.; 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.

Eur J Biochem, 1999 Aug, 263(3), 662 - 70
Nitrile hydratase involved in aldoxime metabolism from Rhodococcus sp . strain YH3-3 purification and characterization; Kato Y et al.; 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.

Appl Microbiol Biotechnol, 1999 Jul, 52(1), 111 - 7
Developments in destructive and non-destructive pathways for selective desulfurizations in oil-biorefining processes
Setti L, Farinelli P, Martino SD, Frassinetti S, Lanzarini G, Pifferi PG.
Biocatalytic desulfurization is still not a commercial technology, but conceptual engineering and sensitivity analyses have shown that the approach is very promising . The purpose of this paper is to investigate further some aspects of the biodesulphurization pathways, discussing the non-destructive pathway with the well-known Rhodococcus rhodochrous IGTS8 . Findings revealed byproducts, such as 2'-hydroxybiphenyl (HBP), sulfite and sulfate, obtained by the desulfurization of dibenzothiophene (DBT), to exert an inhibiting effect . The results suggest that IGTS8 may follow two different metabolic pathways in stationary-growth-phase cells or under growing conditions . The first pathway is characterized by oxidative steps, which convert DBT to DBT sulfoxide and to DBT sulfone . The sulfone is transformed to 2-(2'-hydroxyphenyl)benzene sulfinate and then to HBP and sulfite by a sulfinic acid hydrolase . In the second pathway the sulfone is further oxidized to 2-(2'-hydroxyphenyl)benzene sulfonate and then to HBP and sulfate by a sulfonic acid hydrolase . Experiments using benzene sulfonic acid suggest that the sulfonic acid hydrolase is an induced enzyme.

Appl Microbiol Biotechnol, 1999 Jul, 52(1), 91 - 5
Biosynthesis of polyhydroxyalkanoates from low-rank coal liquefaction products by Pseudomonas oleovorans and Rhodococcus ruber
Fuchtenbusch B, Steinbuchel A.
A screening identified several bacteria that were able to use chemically heterogeneous low-rank coal liquefaction products as complex carbon sources for growth . Pseudomonas oleovorans and Rhodococcus ruber accumulated polyhydroxyalkanoic acids (PHA) amounting to 2%-8% of the cell dry weight when the cells were cultivated on these liquefaction products in the absence of any other carbon source . R . ruber accumulated, in addition to PHA, small amounts of triacylglycerols . The accumulated PHA consisted of 3-hydroxyhexanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate (P . oleovorans) or 3-hydroxybutyric acid and 3-hydroxyvaleric acid (R . ruber) . Low-rank coal liquefaction products obtained from Trichoderma atroviride were better substrates for P . oleovorans than chemically produced fulvic acids.

Appl Microbiol Biotechnol, 1999 Jul, 52(1), 85 - 90
Degradation of alicyclic molecules by Rhodococcus ruber CD4; Schumacher JD et al.; The present work describes investigations on the bacterial degradation of the alicyclic molecule cyclododecane . It represents a structure where the initial degradative steps have to be similar to a "subterminal" attack as there is no "terminal" part of the molecule . We were able to show that the gram-positive bacterium Rhodococcus ruber CD4 DSM 44394 oxidizes cyclododecane to the corresponding alcohol and ketone, the latter being subject to ring fission by a Baeyer-Villiger oxygenase . This key enzyme is an NADPH- and O2-dependent flavoprotein with a substrate specificity for bigger rings . The further metabolism of the resulting lactone gives rise to an omega-hydroxyalkanoic acid that is susceptible to common beta-oxidation . Due to its alicyclic character and its ring size, cyclododecane is comparable to aliphatic bridge components that are an important element in the coal texture . They contribute to the three-dimensional coal structure and thus could serve as a valuable target for the oxidative abilities of R . ruber CD4 to reduce the molecular mass of coal.

Glycobiology, 1999 Sep, 9(9), 957 - 60
Requirement for a different hydrophobic moiety and reliable chromogenic substrate for endo-type glycosylceramidases; Miura Y et al.; A series of synthetic lactosides with aglycones that differed in length and structure were used to determine the substrate specificity of endo-type glycosylceramidases . Endoglycoceramidases (EGCase) from bacteria preferred lactosides with an acylamide structure over simple n-alkyl lactosides . While ceramide glycanase (CGase) from leech did not show preference . N -Acylaminoethyl beta-lactosides and n -alkyl lactosides were substrates for both EGCase and CGase, but N-acylaminobutyl beta-lactosides, whose acylamide residue differs from that in ceramide, were not hydrolyzed by EGCases . Thus, EGCases, but not CGase, appear to require an N-acyl group at the same position as that of intact glycosphingolipid for substrate recognition . A p-nitrophenyl lactoside derivative possessing an N-acyl chain was degraded by both EGCases and CGase and this chromogenic substrate may be an alternative substrate for endo-type glycosylceramidase activity . Km of the chromogenic lactoside for CGase and Rhodococcus EGCase were 28 microM and 2.9 mM, respectively.

Microbiology, 1999 Jul, 145 ( Pt 7), 1721 - 30
Degradation of trichloroethene by a linear-plasmid-encoded alkene monooxygenase in Rhodococcus corallinus (Nocardia corallina) B-276; Saeki H et al.; Rhodococcus corallinus (formerly Nocardia corallina) B-276, isolated with propene as sole carbon and energy source, is able to oxidize trichloroethene (TCE) . Glucose- or propene-grown R . corallinus B-276 cells exhibited no difference in TCE degradation efficiency . TCE degradation was found to be growth-phase-dependent and maximum rates were monitored with stationary-phase cells . K(m) and Vmax values for TCE degradation of R . corallinus B-276 grown in nutrient broth medium in the presence of glucose were 187 microM and 2.4 nmol min-1 (mg protein)-1, respectively . Escherichia coli recombinants harbouring and expressing the alkene monooxygenase genes of R . corallinus B-276 exhibited the ability to degrade TCE . This result provides clear evidence that the alkene monooxygenase of R . corallinus B-276 catalyses TCE oxidation . R . corallinus B-276 was shown to contain four linear plasmids, pNC10 (70 kb), pNC20 (85 kb), pNC30 (185 kb) and pNC40 (235 kb) . The observation that pNC30-deficient strains had lost the ability to grow on propene suggested that the genes of the propene degradation pathway are encoded by the linear plasmid pNC30 . Southern blot analysis with cloned alkene monooxygenase genes from R . corallinus B-276 revealed a positive hybridization signal with the linear plasmid pNC30 . This result clearly shows that the alkene monooxygenase is encoded by the linear plasmid pNC30 . Eleven short-chain-alkene-oxidizing strains were screened for the presence of linear plasmids . Among these, four propene-oxidizing Rhodococcus strains and one ethene-oxidizing Mycobacterium strain were found to contain linear megaplasmids . Southern blot analysis with the alkene monooxygenase revealed positive signals with linear plasmids of two propene-oxidizing Rhodococcus ruber strains . These results indicate that homologous alkene monooxygenases are encoded by linear plasmids in R . ruber strains.

Biochemistry, 1999 Aug 3, 38(31), 9887 - 98
Tertiary and quaternary structures of photoreactive Fe-type nitrile hydratase from Rhodococcus sp . N-771: roles of hydration water molecules in stabilizing the structures and the structural origin of the substrate specificity of the enzyme; Nakasako M et al.; The crystal structure analysis of the Fe-type nitrile hydratase from Rhodococcus sp . N-771 revealed the unique structure of the enzyme composed of the alpha- and beta-subunits and the unprecedented structure of the non-heme iron active center {Nagashima, S., et al . (1998) Nat . Struct . Biol . 5, 347-351} . A number of hydration water molecules were identified both in the interior and at the exterior of the enzyme . The study presented here investigated the roles of the hydration water molecules in stabilizing the tertiary and the quaternary structures of the enzyme, based on the crystal structure and the results from a laser light scattering experiment for the enzyme in solution . Seventy-six hydration water molecules between the two subunits significantly contribute to the alphabeta heterodimer formation by making up the surface shape, forming extensive networks of hydrogen bonds, and moderating the surface charge of the beta-subunit . In particular, 20 hydration water molecules form the extensive networks of hydrogen bonds stabilizing the unique structure of the active center . The amino acid residues hydrogen-bonded to those hydration water molecules are highly conserved among all known nitrile hydratases and even in the homologous enzyme, thiocyanate hydrolase, suggesting the structural conservation of the water molecules in the NHase family . The crystallographic asymmetric unit contained two heterodimers connected by 50 hydration water molecules . The heterotetramer formation in crystallization was clearly explained by the concentration-dependent aggregation state of NHase found in the light scattering measurement . The measurement proved that the dimer-tetramer equilibrium shifted toward the heterotetramer dominant state in the concentration range of 10(-2)-1.0 mg/mL . In the tetramer dominant state, 50 water molecules likely glue the two heterodimers together as observed in the crystal structure . Because NHase exhibits a high abundance in bacterial cells, the result suggests that the heterotetramer is physiologically relevant . In addition, it was revealed that the substrate specificity of this enzyme, recognizing small aliphatic substrates rather than aromatic ones, came from the narrowness of the entrance channel from the bulk solvent to the active center . This finding may give a clue for changing the substrate specificity of the enzyme . Under the crystallization condition described here, one 1,4-dioxane molecule plugged the channel . Through spectroscopic and crystallographic experiments, we found that the molecule prevented the dissociation of the endogenous NO molecule from the active center even when the crystal was exposed to light.

J Appl Microbiol, 1999 Jul, 87(1), 72 - 9
Effect of a mixed culture on co-oxidation during the degradation of saturated hydrocarbon mixture; Ko SH et al.; Two bacterial strains, Pseudomonas aeruginosa K1 and Rhodococcus equi P1, were used to degrade cyclo-alkanes (such as decalin) by a co-oxidation mechanism . Both strains possessed the capacity to degrade a broad range of n-alkane mixtures (C7 to C28) within 24 h of incubation . Strain P1 rapidly degraded 10 gl-1 pristane within 24 h of incubation (mu = 0.36 h-1 and Yx/s = 0.6) . The addition of hexadecane as a growth substrate (above 0.5%, v/v) resulted in complete degradation of 1% (v/v) decalin by strain P1 via a co-oxidation mechanism . Co-oxidation to degrade decalin or pristane by strain K1 proved unsuccessful . Strain P1 was able to degrade decalin totally in a saturated hydrocarbon mixture . Strain K1 was only able to degrade hexadecane from the hydrocarbon mixture, but its degradation rate was higher than that of strain P1 . Therefore, there was competition for the hexadecane needed to co-oxidize decalin . As a result, degradation of the hydrocarbon mixture, especially decalin, was incomplete in a mixed culture of strain P1 and K1 . Serial addition of hexadecane (twice) allowed complete degradation of the remaining decalin by strain P1 . Also, the biodegradation rate of the hydrocarbon mixture by a microbial population from gasoline-contaminated soil was delayed by addition of strain K1 to the population, while the addition of strain P1 resulted in an increase in the biodegradation rate.

Antimicrob Agents Chemother, 1999 Aug, 43(8), 2093 - 6
In vitro activities of polycationic peptides alone and in combination with clinically used antimicrobial agents against Rhodococcus equi; Giacometti A et al.; The in vitro activities of magainin II, nisin, and ranalexin alone and in combination with other antimicrobial agents against six clinical isolates of Rhodococcus equi were investigated by MIC and time-kill studies . All isolates were more susceptible to nisin . A positive interaction was observed when the peptides were combined with ampicillin, ceftriaxone, rifabutin, rifampin, azithromycin, clarithromycin, and vancomycin.

Biochemistry (Mosc), 1999 Jul, 64(7), 824 - 31
Isolation and characterization of catechol 1,2-dioxygenases from Rhodococcus rhodnii strain 135 and Rhodococcus rhodochrous strain 89: comparison with analogous enzymes of the ordinary and modified ortho-cleavage pathways; Solyanikova IP et al.; Catechol 1,2-dioxygenases of the ordinary ortho-cleavage pathway have been isolated from strains Rhodococcus rhodnii 135 and Rhodococcus rhodochrous 89 grown on phenol as the sole source of carbon and energy . The activities of the catechol 1,2-dioxygenases with 3- and 4-methylpyrocatechols were 1.3-1.5 times higher than those with pyrocatechol . The rate of oxidation of 3-chloropyrocatechol catalyzed by both enzymes was 20% of the rate of oxidation of unsubstituted pyrocatechol . The enzymes are homodimers composed of 37-kD subunits.

New Microbiol, 1999 Jul, 22(3), 249 - 56
Characterization of antarctic hydrocarbon-degrading bacteria capable of producing bioemulsifiers; Yakimov MM et al.; During screening for biosurfactant-producing, n-alkane-degrading marine bacteria, two heterotrophic bacterial strains were isolated from enriched mixed cultures, obtained from Terra Nova Bay (Ross sea, Antarctica) by using aliphatic and artomatic hydrocarbons as the principal carbon source . These gram-positive, aerobic, cocci-shaped bacteria use a various number of organic compounds, including aliphatic hydrocarbons, volatile fatty acids, and biphenyl . During cultivation on n-alkanes as sole source of carbon and energy, all strains produced both an extracellular and cell-bound surface-active mixture of trehalose lipids which reduced the surface tension of water from 72 mN/m to 32mN/m . This class of glycolipids was found to be produced only by marine rhodococci . The 16S-rRNA gene sequence analysis showed that both strains are members of the G + C rich gram-positive group of the phylum Proteobacteria and was found to be almost identical to that of Rhodococcus fascians DSM 20669 . The potential of these strains for in situ bioremediation of contaminated cold marine environment is discussed in the present study.

Appl Microbiol Biotechnol, 1999 Jun, 51(6), 786 - 93
Isolation and characterization of indene bioconversion genes from Rhodococcus strain I24; Treadway SL et al.; Rhodococcus strain 124 is able to convert indene into indandiol via the actions of at least two dioxygenase systems and a putative monooxygenase system . We have identified a cosmid clone from 124 genomic DNA that is able to confer the ability to convert indene to indandiol upon Rhodococcus erythropolis SQ1, a strain that normally can not convert or metabolize indene . HPLC analysis reveals that the transformed SQ1 strain produces cis-(1R,2S)-indandiol, suggesting that the cosmid clone encodes a naphthalenetype dioxygenase . DNA sequence analysis of a portion of this clone confirmed the presence of genes for the dioxygenase as well as genes encoding a dehydrogenase and putative aldolase . These genes will be useful for manipulating indene bioconversion in Rhodococcus strain 124.

Vet Pathol, 1999 Jul, 36(4), 336 - 9
Disseminated Rhodococcus equi infection in two goats; Davis WP et al.; Rhodococcus equi infection was diagnosed in two goats from the same herd . At necropsy, numerous caseating granulomas were disseminated throughout the liver, lungs, abdominal lymph nodes, medulla of right humerus, and the right fifth rib of goat No . 1, and the liver of goat No . 2 . Histopathologic examination confirmed the presence of multiple caseating granulomas in these organs . Numerous gram-positive and Giemsa-positive coccobacilli were identified within the cytoplasm of macrophages . Aerobic bacterial cultures of the liver and lung from both goats yielded a pure growth of R . equi . R . equi antigens were immunohistochemically identified in caseating granulomas from both goats . However, the 15- to 17-kd virulence antigens of R . equi were not detected, suggesting possible infection by an avirulent strain of this organism.

Eur J Clin Microbiol Infect Dis, 1999 May, 18(5), 324 - 9
Etiology, clinical features and outcome of splenic microabscesses in HIV-infected patients with prolonged fever; Bernabeu-Wittel M et al.; A prospective study was conducted to determine the etiology, clinical features, and outcome in a series of 32 consecutively enrolled HIV-infected patients with prolonged fever in whom high resolution (7.5 Mhz) sonography revealed multiple splenic microabscesses . Conventional (3.5 Mhz) sonography showed no splenic abnormalities in any patients . The diagnoses were: tuberculosis (14), visceral leishmaniasis (7), disseminated Mycobacterium avium complex infection (5), Salmonella spp . bacteremia (2), lymphoma (2), disseminated Rhodococcus equi infection (1), disseminated Candida krusei infection (1) and Pneumocystis carinii pneumonia (1) . Twenty-eight patients were followed up for six months and four were lost to follow-up . In 16 patients with a clinical cure and microbiological eradication, the findings on follow-up high resolution sonography were normal, and in two patients the microabscesses persisted; ten patients died . In conclusion, the findings suggest splenic microabscesses may be a frequent condition in HIV-infected patients with prolonged fever, being an unspecific manifestation of the opportunistic diseases causing fever of unknown origin in this population . They cannot be detected by conventional abdominal sonography, whereas high resolution sonography is a useful technique for their detection and follow-up.

Mol Microbiol, 1999 Aug, 33(3), 510 - 23
The smcL gene of Listeria ivanovii encodes a sphingomyelinase C that mediates bacterial escape from the phagocytic vacuole; Gonzalez-Zorn B et al.; The ruminant pathogen Listeria ivanovii differs from Listeria monocytogenes in that it causes strong, bizonal haemolysis and a characteristic shovel-shaped co-operative haemolytic ('CAMP-like') reaction with Rhodococcus equi . We cloned the gene responsible for the differential haemolytic properties of L . ivanovii, smcL . It encodes a sphingomyelinase C (SMase) highly similar (> 50% identity) to the SMases from Staphylococcus aureus (beta-toxin), Bacillus cereus and Leptospira interrogans . smcL was transcribed monocistronically and was expressed independently of PrfA . Low-stringency Southern blots demonstrated that, within the genus Listeria, smcL was present only in L . ivanovii . We constructed an smcL knock-out mutant . Its phenotype on blood agar was identical to that of L . monocytogenes (i.e . weak haemolysis and no shovel-shaped CAMP-like reaction with R . equi ) . This mutant was less virulent for mice, and its intracellular proliferation was impaired in the bovine epithelial-like cell line MDBK . The role of SmcL in intracellular survival was investigated using an L . monocytogenes mutant lacking the membrane-damaging determinants hly, plcA and plcB, being thus unable to grow intracellularly . Complementation of this mutant with smcL on a plasmid was sufficient to promote bacterial intracellular proliferation in MDBK cells . Transmission electron microscopy showed that SmcL mediates the disruption of the phagocytic vacuole and the release of bacteria into the cytosol . Therefore, L . ivanovii possesses a third phospholipase with membrane-damaging activity that, together with PlcA and PlcB, may act in concert with the pore-forming toxin Hly to mediate efficient escape from the vacuolar compartment . The 5' end of smcL is contiguous with the internalin locus i-inlFE, which is also specific to L . ivanovii and is required for full virulence in mice . Thus, smcL forms part of a novel virulence gene cluster in Listeria that is species specific.

Appl Environ Microbiol, 1999 Jul, 65(7), 2961 - 8
Physiological adaptations involved in alkane assimilation at a low temperature by Rhodococcus sp . strain Q15; Whyte LG et al.; We examined physiological adaptations which allow the psychrotroph Rhodococcus sp . strain Q15 to assimilate alkanes at a low temperature (alkanes are contaminants which are generally insoluble and/or solid at low temperatures) . During growth at 5 degrees C on hexadecane or diesel fuel, strain Q15 produced a cell surface-associated biosurfactant(s) and, compared to glucose-acetate-grown cells, exhibited increased cell surface hydrophobicity . A transmission electron microscopy examination of strain Q15 grown at 5 degrees C revealed the presence of intracellular electron-transparent inclusions and flocs of cells connected by an extracellular polymeric substance (EPS) when cells were grown on a hydrocarbon and morphological differences between the EPS of glucose-acetate-grown and diesel fuel-grown cells . A lectin binding analysis performed by using confocal scanning laser microscopy (CSLM) showed that the EPS contained a complex mixture of glycoconjugates, depending on both the growth temperature and the carbon source . Two glycoconjugates {beta-D-Gal-(1-3)-D-GlcNAc and alpha-L-fucose} were detected only on the surfaces of cells grown on diesel fuel at 5 degrees C . Using scanning electron microscopy, we observed strain Q15 cells on the surfaces of octacosane crystals, and using CSLM, we observed strain Q15 cells covering the surfaces of diesel fuel microdroplets; these findings indicate that this organism assimilates both solid and liquid alkane substrates at a low temperature by adhering to the alkane phase . Membrane fatty acid analysis demonstrated that strain Q15 adapted to growth at a low temperature by decreasing the degree of saturation of membrane lipid fatty acids, but it did so to a lesser extent when it was grown on hydrocarbons at 5 degrees C; these findings suggest that strain Q15 modulates membrane fluidity in response to the counteracting influences of low temperature and hydrocarbon toxicity.

Biochem Biophys Res Commun, 1999 Jun 24, 260(1), 89 - 93
The Glu residue in the conserved Asn-Glu-Pro sequence of endoglycoceramidase is essential for enzymatic activity; Sakaguchi K et al.; Endoglycoceramidase (EGCase) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids . We previously cloned the gene encoding EGCase II of Rhodococcus sp . M-777 and reported that the deduced amino acid sequence contained the Asn-Glu-Pro (NEP) sequence, conserved as part of the active site of family A cellulases (endo-1,4-beta-glucanases) (J . Biol . Chem . 272, 19846, 1997) . The NEP sequence was also found in the deduced amino acid sequence of the newly cloned EGCase gene of Rhodococcus sp . C9 . Replacement of the Glu residue in the NEP sequence with Gln or Asp by site-directed mutagenesis caused marked loss of enzymatic activity in both the M-777 and C9 EGCases but did not affect the expression of EGCase protein . This result clearly indicated that the NEP sequence is part of the active site of EGCase, in which the Glu residue plays an important role in the catalytic reaction, possibly in the same manner as in endo-1,4-beta-glucanase .

Infect Immun, 1999 Jul, 67(7), 3548 - 57
Role of the 85-kilobase plasmid and plasmid-encoded virulence-associated protein A in intracellular survival and virulence of Rhodococcus equi; Giguere S et al.; Rhodococcus equi is a facultative intracellular pathogen of macrophages and a cause of pneumonia in young horses (foals) and immunocompromised people . Isolates of R . equi from pneumonic foals typically contain large, 85- or 90-kb plasmids encoding a highly immunogenic virulence-associated protein (VapA) . The objective of this study was to determine the role of the 85-kb plasmid and VapA in the intracellular survival and virulence of R . equi . Clinical isolates containing the plasmid and expressing VapA efficiently replicated within mouse macrophages in vitro, while plasmid-cured derivatives of these organisms did not multiply intracellularly . An isolate harboring the large plasmid also replicated in the tissues of experimentally infected mice, whereas its plasmid-cured derivative was rapidly cleared . All foals experimentally infected with a plasmid-containing clinical isolate developed severe bronchopneumonia, whereas the foals infected with its plasmid-cured derivative remained asymptomatic and free of visible lung lesions . By day 14 postinfection, lung bacterial burdens had increased considerably in foals challenged with the plasmid-containing clinical isolate . In contrast, bacteria could no longer be cultured from the lungs of foals challenged with the isogenic plasmid-cured derivative . A recombinant, plasmid-cured derivative expressing wild-type levels of VapA failed to replicate in macrophages and remained avirulent for both mice and foals . These results show that the 85-kb plasmid of R . equi is essential for intracellular replication within macrophages and for development of disease in the native host, the foal . However, expression of VapA alone is not sufficient to restore the virulence phenotype.

Arch Microbiol, 1999 May-Jun, 171(6), 430 - 8
Linear alkanesulfonates as carbon and energy sources for gram-positive and gram-negative bacteria; Reichenbecher W et al.; Several bacteria from soil and rainwater samples were enriched and isolated with propanesulfonate or butanesulfonate as sole carbon and energy source . Most of the strains isolated utilized nonsubstituted alkanesulfonates with a chain length of C3-C6 and the substituted sulfonates taurine and isethionate as carbon and energy source . A gram-positive isolate, P40, and a gram-negative isolate, P53, were characterized in more detail . Phylogenetic analysis grouped strain P40 within group IV of the genus Rhodococcus and showed a close relationship with Rhodococcus opacus . After phylogenetic and physiological analyses, strain P53 was identified as Comamonas acidovorans . Both bacteria also utilized a wide range of sulfonates as sulfur source . Strain P40, but not strain P53, released sulfite into the medium during dissimilation of sulfonated compounds . Cell-free extracts of strain P53 exhibited high sulfite oxidase activity {2.34 U (mg protein)-1} when assayed with ferricyanide, but not with cytochrome c . Experiments with whole-cell suspensions of both strains showed that the ability to dissimilate 1-propanesulfonate was specifically induced during growth on this substrate and was not present in cells grown on propanol, isethionate or taurine . Whole-cell suspensions of both strains accumulated acetone when oxidizing the non-growth substrate 2-propanesulfonate . Strain P40 cells also accumulated sulfite under these conditions . Stoichiometric measurements with 2-propanesulfonate as substrate in oxygen electrode experiments indicate that the nonsubstituted alkanesulfonates were degraded by a monooxygenase . When strain P53 grew with nonsubstituted alkanesulfonates as carbon and energy source, cells expressed high amounts of yellow pigments, supporting the proposition that an oxygenase containing iron sulfur centres or flavins was involved in their degradation.

J Vet Intern Med, 1999 May-Jun, 13(3), 206 - 12
Peripheral blood lymphocyte subpopulations and immunoglobulin concentrations in healthy foals and foals with Rhodococcus equi pneumonia; Flaminio MJ et al.; Infectious diseases are common in foals aged 1-5 months . The objectives of this investigation were to evaluate immunologic parameters in foals from birth to weaning to establish reference values for the proportion of circulating lymphocytes that were helper (CD4+) or cytotoxic (CD8+) T cells, or B cells; to measure serum immunoglobulin (IgM and IgG) concentrations; and to compare these immunologic parameters to values in foals with naturally occurring Rhodococcus equi pneumonia and in adult horses . Peripheral blood lymphocyte subpopulations were determined by flow cytometric analysis, and serum IgG and IgM concentrations were determined by radial immunodiffusion . Flow cytometric analysis of lymphocyte subpopulations suggested age-related changes in the cell-mediated immune system in horses . Absolute circulating CD4+ and CD8+ T lymphocytes and B cells increased linearly up to 3 months of age . Circulating B cell concentrations from birth to 6 months of age were greater than values in adult horses and the lymphocyte differences among the age groups are mainly due to variation in B lymphocytes . Both absolute and proportional B cell concentrations were greater in foals with R equi pneumonia than in healthy foals at the same age . The increase in absolute cell counts of each subpopulation was dependent on the increase of absolute peripheral blood lymphocyte count . Serum IgG concentration increased linearly from 1 to 3 months of age, and serum IgM concentrations increased from 1 to 6 months of age . These data suggest age-dependent cell-mediated and humoral development in young foals.

Trends Biotechnol, 1999 Jun, 17(6), 244 - 8
An enzyme controlled by light: the molecular mechanism of photoreactivity in nitrile hydratase; Endo I et al.; Extensive studies have revealed the molecular mechanism of the photoreactivity of nitrile hydratase from Rhodococcus sp . N-771 . In the inactive enzyme, nitric oxide is bound to the non-heme ferric iron at the catalytic center, stabilized by a claw-like structure formed by two post-translationally modified cysteines and a serine . The inactive nitrile hydratase is activated by the photoinduced release of the nitric oxide . This result might provide a means of designing novel photoreactive chemical compounds or proteins that would be applicable to biochips and light-controlled metabolic systems.

J Appl Microbiol, 1999 May, 86(5), 752 - 60
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; Bigey F et al.; 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.

J Clin Pathol, 1999 Jan, 52(1), 68 - 71
Infection by Rhodococcus equi in a patient with AIDS: histological appearance mimicking Whipple's disease and Mycobacterium avium-intracellulare infection; Hamrock D et al.; Rhodococcus equi pneumonia with systemic dissemination is being reported increasingly in immunocompromised patients . This is the first case report of disseminated R equi infection with biopsy documented involvement of the large intestine . The patient was a 46 year old male with AIDS who was diagnosed with cavitating pneumonia involving the left lower lobe . R equi was isolated in culture from the blood and lung biopsies . Subsequently, the patient developed anaemia, diarrhoea, and occult blood in the stool . Colonoscopy revealed several colonic polyps . Histological examination of the colon biopsies showed extensive submucosal histiocytic infiltration with numerous Gram positive coccobacilli and PAS positive material in the histiocytes . Electron microscopy showed variably shaped intrahistiocytic organisms which were morphologically consistent with R equi in the specimen . Disseminated R equi infection may involve the lower gastrointestinal tract and produce inflammatory polyps with foamy macrophages which histologically resemble those seen in Whipple's disease and Mycobacterium avium-intracellulare infection.

FEMS Immunol Med Microbiol, 1999 May, 24(1), 1 - 9
Live virulent Rhodococcus equi, rather than killed or avirulent, elicits protective immunity to R . equi infection in mice; Takai S et al.; Mice inoculated intravenously with a sublethal dose of live virulent Rhodococcus equi ATCC 33701 that contained an 85-kb virulence plasmid were immune to a lethal intravenous challenge of ATCC 33701 . This immunity depended upon the dose of immunization and developed rapidly: mice primed with 10(5) live ATCC 33701 eliminated the challenged bacteria more rapidly than mice primed with doses ranging from 10(2) to 10(4) bacteria, and mice given 10(5) live ATCC 33701 intravenously withstood the lethal challenge as early as 5 days after the initial inoculation . However, this protective immunity did not develop in mice immunized with doses of heat-killed ATCC 33701 ranging from 10(6) to 10(8), or in mice immunized with doses of live ATCC 33701P-, a plasmid-cured derivative (avirulent), in doses ranging from 10(5) to 10(7) . These mice had positive antibody titers against R . equi at the challenge (14 days after priming) . Adoptive transfer of resistance to virulent R equi was obtained with spleen cells from mice immunized with live ATCC 33701, but not monoclonal antibody to 15- to 17-kDa virulence-associated antigens . These results revealed that live ATCC 33701P-, a plasmid-cured derivative of virulent R equi, could not elicit protective immunity, and are consistent with previous observations that protective immunity was induced by live virulent, but not killed organisms.

Biodegradation, 1998, 9(6), 475 - 86
19F NMR study on the biodegradation of fluorophenols by various Rhodococcus species; Bondar VS et al.; Of all NMR observable isotopes 19F is the one perhaps most convenient for studies on biodegradation of environmental pollutants . The reasons underlying this potential of 19F NMR are discussed and illustrated on the basis of a study on the biodegradation of fluorophenols by four Rhodococcus strains . The results indicate marked differences between the biodegradation pathways of fluorophenols among the various Rhodococcus species . This holds not only for the level and nature of the fluorinated biodegradation pathway intermediates that accumulate, but also for the regioselectivity of the initial hydroxylation step . Several of the Rhodococcus species contain a phenol hydroxylase that catalyses the oxidative defluorination of ortho-fluorinated di- and trifluorophenols . Furthermore, it is illustrated how the 19F NMR technique can be used as a tool in the process of identification of an accumulated unknown metabolite, in this case most likely 5-fluoromaleylacetate . Altogether, the 19F NMR technique proved valid to obtain detailed information on the microbial biodegradation pathways of fluorinated organics, but also to provide information on the specificity of enzymes generally considered unstable and, for this reason, not much studied so far.

Biotechnol Appl Biochem, 1999 Jun, 29 ( Pt 3), 217 - 21
Production of cholesterol oxidase by Rhodococcus equi No . 23 in a jar fermenter; Chou CC et al.; Rhodococcus equi No . 23 was grown in a batch fermenter . The effects of cultivation temperature, pH of the culture medium, aeration rate and agitation speed on the production of cholesterol oxidase (CholOx) by the test organism were examined . Results revealed that the cultivation temperature, the pH of the medium, the aeration rate and the agitation speed all affected the production of CholOx by R . equi No . 23 . Adjusting the operation variables during the cultivation period increased the production of CholOx effectively and prevented the occurrence of overflow of foam during the fermentation period . A maximum CholOx activity of 0.34 unit/ml with a volumetric production rate of 0.011 unit/h per ml could be achieved in 30 h of cultivation at an aeration rate of 5.0 l/min, if the pH of the culture medium, the cultivation temperature and the agitation speed were controlled at 6.5, 39 degrees C and 200 rev . /min respectively during the first 24 h of cultivation, then shifted to 7.5, 37 degrees C and 300 rev./min respectively.

J Clin Microbiol, 1999 Jun, 37(6), 1971 - 6
Distribution of a Nocardia brasiliensis catalase gene fragment in members of the genera Nocardia, Gordona, and Rhodococcus; Vera-Cabrera L et al.; An immunodominant protein from Nocardia brasiliensis, P61, was subjected to amino-terminal and internal sequence analysis . Three sequences of 22, 17, and 38 residues, respectively, were obtained and compared with the protein database from GenBank by using the BLAST system . The sequences showed homology to some eukaryotic catalases and to a bromoperoxidase-catalase from Streptomyces violaceus . Its identity as a catalase was confirmed by analysis of its enzymatic activity on H2O2 and by a double-staining method on a nondenaturing polyacrylamide gel with 3,3'-diaminobenzidine and ferricyanide; the result showed only catalase activity, but no peroxidase . By using one of the internal amino acid sequences and a consensus catalase motif (VGNNTP), we were able to design a PCR assay that generated a 500-bp PCR product . The amplicon was analyzed, and the nucleotide sequence was compared to the GenBank database with the observation of high homology to other bacterial and eukaryotic catalases . A PCR assay based on this target sequence was performed with primers NB10 and NB11 to confirm the presence of the NB10-NB11 gene fragment in several N . brasiliensis strains isolated from mycetoma . The same assay was used to determine whether there were homologous sequences in several type strains from the genera Nocardia, Rhodococcus, Gordona, and Streptomyces . All of the N . brasiliensis strains presented a positive result but only some of the actinomycetes species tested were positive in the PCR assay . In order to confirm these findings, genomic DNA was subjected to Southern blot analysis . A 1.7-kbp band was observed in the N . brasiliensis strains, and bands of different molecular weight were observed in cross-reacting actinomycetes . Sequence analysis of the amplicons of selected actinomycetes showed high homology in this catalase fragment, thus demonstrating that this protein is highly conserved in this group of bacteria.

Immunology, 1999 Jan, 96(1), 122 - 7
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; Kasuga-Aoki H et al.; 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.

Immunology, 1999 Jan, 96(1), 10 - 5
Movement disorders in encephalitis induced by Rhodococcus aurantiacus infection relieved by the administration of L-dopa and anti-T-cell antibodies; Min Y et al.; 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.

Biochemistry, 1999 May 4, 38(18), 5772 - 8
Haloalkane dehalogenases: steady-state kinetics and halide inhibition; Schindler JF et al.; The substrate specificities and product inhibition patterns of haloalkane dehalogenases from Xanthobacter autotrophicus GJ10 (XaDHL) and Rhodococcus rhodochrous (RrDHL) have been compared using a pH-indicator dye assay . In contrast to XaDHL, RrDHL is efficient toward secondary alkyl halides . Using steady-state kinetics, we have shown that halides are uncompetitive inhibitors of XaDHL with 1, 2-dichloroethane as the varied substrate at pH 8.2 (Cl-, Kii = 19 +/- 0.91; Br-, Kii = 2.5 +/- 0.19 mM; I-, Kii = 4.1 +/- 0.43 mM) . Because they are uncompetitive with the substrate, halide ions do not bind to the free form of the enzyme; therefore, halide ions cannot be the last product released from the enzyme . The Kii for chloride was pH dependent and decreased more than 20-fold from 61 mM at pH 8.9 to 2.9 mM at pH 6.5 . The pH dependence of 1/Kii showed simple titration behavior that fit to a pKa of approximately 7.5 . The kcat was maximal at pH 8.2 and decreased at lower pH . A titration of kcat versus pH also fits to a pKa of approximately 7.5 . Taken together, these data suggest that chloride binding and kcat are affected by the same ionizable group, likely the imidazole of a histidyl residue . In contrast, halides do not inhibit RrDHL . The Rhodococcus enzyme does not contain a tryptophan corresponding to W175 of XaDHL, which has been implicated in halide ion binding . The site-directed mutants W175F and W175Y of XaDHL were prepared and tested for halide ion inhibition . Halides do not inhibit either W175F or W175Y XaDHL.

Appl Environ Microbiol, 1999 May, 65(5), 2092 - 102
Rhodococcus erythropolis DCL14 contains a novel degradation pathway for limonene; van der Werf MJ et al.; Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample . This organism was identified as a strain of Rhodococcus erythropolis by chemotaxonomic and genetic studies . R . erythropolis DCL14 also assimilated the terpenes limonene-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (-)-menthol, while perillyl alcohol was not utilized as a carbon and energy source . Induction tests with cells grown on limonene revealed that the oxygen consumption rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high . Limonene-induced cells of R . erythropolis DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1, 2-monooxygenase activity, a cofactor-independent limonene-1, 2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity . Product accumulation studies showed that (1S,2S,4R)-limonene-1,2-diol, (1S, 4R)-1-hydroxy-2-oxolimonene, and (3R)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)-limonene degradation pathway . The opposite enantiomers {(1R,2R,4S)-limonene-1,2-diol, (1R, 4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxoheptanoate} were found in the (4S)-limonene degradation pathway, while accumulation of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed . These results show that R . erythropolis DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide . This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone . This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate . In the presence of coenzyme A and ATP this acid is converted further, and this finding, together with the high levels of isocitrate lyase activity in extracts of limonene-grown cells, suggests that further degradation takes place via the beta-oxidation pathway.

J Bacteriol, 1999 May, 181(9), 2752 - 8
Evidence for an inducible nucleotide-dependent acetone carboxylase in Rhodococcus rhodochrous B276; Clark DD et al.; The metabolism of acetone was investigated in the actinomycete Rhodococcus rhodochrous (formerly Nocardia corallina) B276 . Suspensions of acetone- and isopropanol-grown R . rhodochrous readily metabolized acetone . In contrast, R . rhodochrous cells cultured with glucose as the carbon source lacked the ability to metabolize acetone at the onset of the assay but gained the ability to do so in a time-dependent fashion . Chloramphenicol and rifampin prevented the time-dependent increase in this activity . Acetone metabolism by R . rhodochrous was CO2 dependent, and 14CO2 fixation occurred concomitant with this process . A nucleotide-dependent acetone carboxylase was partially purified from cell extracts of acetone-grown R . rhodochrous by DEAE-Sepharose chromatography . Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the acetone carboxylase was composed of three subunits with apparent molecular masses of 85, 74, and 16 kDa . Acetone metabolism by the partially purified enzyme was dependent on the presence of a divalent metal and a nucleoside triphosphate . GTP and ITP supported the highest rates of acetone carboxylation, while CTP, UTP, and XTP supported carboxylation at 10 to 50% of these rates . ATP did not support acetone carboxylation . Acetoacetate was determined to be the stoichiometric product of acetone carboxylation . The longer-chain ketones butanone, 2-pentanone, 3-pentanone, and 2-hexanone were substrates . This work has identified an acetone carboxylase with a novel nucleotide usage and broader substrate specificity compared to other such enzymes studied to date . These results strengthen the proposal that carboxylation is a common strategy used for acetone catabolism in aerobic acetone-oxidizing bacteria.

Microbiology, 1999 Mar, 145 ( Pt 3), 561 - 8
Characterization of IS2112, a new insertion sequence from Rhodococcus, and its relationship with mobile elements belonging to the IS110 family; Kulakov LA et al.; A new insertion sequence (IS2112) was identified in the genome of the 1-haloalkane-utilizing bacterium Rhodococcus rhodochrous NCIMB 13064 . The insertion element is 1415 bp long, does not contain terminal inverted repeats, and is not flanked by directly repeated sequences . IS2112 belongs to the IS110 family of transposable elements, and forms a separate subfamily, along with IS116 . Two copies of IS2112 were found in R . rhodochrous NCIMB 13064 and one, two or three copies of a similar sequence were detected in five other 1-haloalkane-degrading Rhodococcus strains . There were no sequences homologous to IS2112 found in the 1-haloalkane-degrading 'Pseudomonas pavonaceae' 170 and Rhodococcus sp . HA1 or in several Rhodococcus strains which do not utilize haloalkanes . IS2112 was originally found in plasmid pRTL1 of R . rhodochrous NCIMB 13064, which harbours genes encoding utilization of 1-haloalkanes, and was located 5 kbp upstream of the haloalkane dehalogenase gene (dhaA) . Although the second copy of IS2112 in strain NCIMB 13064 was also present on the pRTL1 plasmid, these sequences do not apparently comprise a single composite transposon encoding haloalkane utilization . An analysis of derivatives of NCIMB 13064 revealed that IS2112 was involved in genome rearrangements . IS2112 appeared to change its location as a result of transposition and as a result of other rearrangements of the NCIMB 13064 genome.

Am J Vet Res, 1999 Apr, 60(4), 414 - 9
Comparison of microbiologic and high-performance liquid chromatography assays to determine plasma concentrations, pharmacokinetics, and bioavailability of erythromycin base in plasma of foals after intravenous or intragastric administration; Lakritz J et al.; OBJECTIVE: To determine pharmacokinetics and bioavailability of erythromycin base after intragastric administration and erythromycin lactobionate after IV administration to healthy foals and to compare a microbiologic assay with a high-performance liquid chromatography (HPLC) method to determine plasma concentrations of erythromycin A . ANIMALS: 6 healthy foals that were 2 to 4 months old . PROCEDURE: Foals were given single doses of erythromycin (10 mg/kg of body weight, IV, and 25 mg/kg, intragastrically) in a crossover study . Venous blood samples were obtained at specific times after drug administration, and plasma was harvested for determination of erythromycin concentrations by microbiologic assay and a HPLC method Pharmacokinetic analysis of plasma concentration-time data was performed, and results derived from each method were compared . RESULTS: Concentration-time profiles for IV administration were best described by a two-compartment open model . Comparing pharmacokinetic data obtained by the 2 methods revealed substantial differences in results . Values for area under the plasma concentration-time curve and area under the first moment of the curve were substantially higher when determined by the bioassay, indicating overestimation of plasma concentration-time data by this method . The derived rate transfer constants (K21 and K(e)1) and mean residence time were significantly different, when determined by the bioassay . Systemic bioavailability of erythromycin base was low in all foals . CONCLUSIONS AND CLINICAL RELEVANCE: The bioassay method overestimated plasma concentrations of erythromycin, compared with the HPLC method . Despite low systemic bioavailability of erythromycin base administered intragastrically, plasma concentrations of erythromycin exceeded, for at least 4 hours, the minimum inhibitory concentration of most Rhodococcus equi isolates.

Biodegradation, 1998, 9(5), 381 - 7
Influence of selected physical parameters on the biodegradation of acrylamide by immobilized cells of Rhodococcus sp; Nawaz MS et al.; The influences of concentration of acrylamide, pH, temperature, duration of storage of encapsulated cells and presence of different metals and chelators on the ability of immobilized cells of a Rhodococcus sp . to degrade acrylamide were evaluated . Immobilized cells (3 g) rapidly degraded 64 and 128 mM acrylamide in 3 and 5 h, respectively, whereas free cells took more than 24 h to degrade 64 mM acrylamide . An acrylamide concentration of 128 mM inhibited the growth of the free cells . Immobilized bacteria were slow to degrade acrylamide at 10 degrees C . Less than 60% of acrylamide was degraded in 4 h . However, 100% of the compound was degraded in less than 3 h at 28 degrees C and 45 degrees C . The optimum pH for the degradation of acrylamide by encapsulated cells was pH 7.0 . Less than 10% of acrylamide was degraded at pH 6.0, while ca . 60% of acrylamide was degraded at pH 8.0 and 8.5 . Copper and nickel inhibited the degradation, suggesting the presence of sulfhydryl (-SH) groups in the active sites of the acrylamide degrading amidase . Iron enhanced the rates of degradation and chelators (EDTA and 1,10 phenanthroline) reduced the rates of degradation suggesting the involvement of iron in its active site(s) of the acrylamide-degrading-amidase . Immobilized cells could be stored up to 10 days without any detectable loss of acrylamide-degrading activity.

Appl Environ Microbiol, 1999 Apr, 65(4), 1589 - 95
The alkene monooxygenase from Xanthobacter strain Py2 is closely related to aromatic monooxygenases and catalyzes aromatic monohydroxylation of benzene, toluene, and phenol; Zhou NY et al.; The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter strain Py2 (Xamo) have been located on a 4.9-kb fragment of chromosomal DNA previously cloned in cosmid pNY2 . Sequencing and analysis of the predicted amino acid sequences indicate that the components of Xamo are homologous to those of the aromatic monooxygenases, toluene 2-, 3-, and 4-monooxygenase and benzene monooxygenase, and that the gene order is identical . The genes and predicted polypeptides are aamA, encoding the 497-residue oxygenase alpha-subunit (XamoA); aamB, encoding the 88-residue oxygenase gamma-subunit (XamoB); aamC, encoding the 122-residue ferredoxin (XamoC); aamD, encoding the 101-residue coupling or effector protein (XamoD); aamE, encoding the 341-residue oxygenase beta-subunit (XamoE); and aamF, encoding the 327-residue reductase (XamoF) . A sequence with >60% concurrence with the consensus sequence of sigma54 (RpoN)-dependent promoters was identified upstream of the aamA gene . Detailed comparison of XamoA with the oxygenase alpha-subunits from aromatic monooxygenases, phenol hydroxylases, methane monooxygenase, and the alkene monooxygenase from Rhodococcus rhodochrous B276 showed that, despite the overall similarity to the aromatic monooxygenases, XamoA has some distinctive characteristics of the oxygenases which oxidize aliphatic, and particularly alkene, substrates . On the basis of the similarity between Xamo and the aromatic monooxygenases, Xanthobacter strain Py2 was tested and shown to oxidize benzene, toluene, and phenol, while the alkene monooxygenase-negative mutants NZ1 and NZ2 did not . Benzene was oxidized to phenol, which accumulated transiently before being further oxidized . Toluene was oxidized to a mixture of o-, m-, and p-cresols (39.8, 18, and 41.7%, respectively) and a small amount (0.5%) of benzyl alcohol, none of which were further oxidized . In growth studies Xanthobacter strain Py2 was found to grow on phenol and catechol but not on benzene or toluene; growth on phenol required a functional alkene monooxygenase . However, there is no evidence of genes encoding steps in the metabolism of catechol in the vicinity of the aam gene cluster . This suggests that the inducer specificity of the alkene monooxygenase may have evolved to benefit from the naturally broad substrate specificity of this class of monooxygenase and the ability of the host strain to grow on catechol.

Biosens Bioelectron, 1999 Feb, 14(2), 187 - 93
Development of an automated microbial sensor system; Heim S et al.; An automated whole cell biosensor system was developed by integration of immobilized microbial cells in a flow-through system with screen-printed flow-through electrodes as detectors . The detectors used were thick-film Pt-electrodes in a 3-electrode configuration constructed as sandwich flow-through cells with a volume of about 36 microliters polarized at -900 mV . The measuring principle was the determination of oxygen consumption due to the microbial metabolism . Fructose was used as model analyte . The microorganisms were immobilized on cellulose-acetate membranes and integrated into a newly created reaction chamber (membrane reactor) . The microbial cells used were Rhodococcus erythropolis and Issatchenkia orientalis known to be suitable for the determination of biological oxygen demand.

J Biochem (Tokyo), 1999 Apr, 125(4), 696 - 704
Functional expression of nitrile hydratase in Escherichia coli: requirement of a nitrile hydratase activator and post-translational modification of a ligand cysteine; Nojiri M et al.; The nitrile hydratase (NHase) from Rhodococcus sp . N-771 is a photoreactive enzyme that is inactivated on nitrosylation of the non-heme iron center and activated on photo-dissociation of nitric oxide (NO) . The nitrile hydratase operon consists of six genes encoding NHase regulator 2, NHase regulator 1, amidase, NHase alpha subunit, NHase beta subunit and NHase activator . We overproduced the NHase in Escherichia coli using a T7 expression system . The NHase was functionally expressed in E . coli only when the NHase activator encoded downstream of the beta subunit gene was co-expressed and the transformant was grown at 30 degrees C or less . A ligand cysteine, alphaCys112, of the recombinant NHase was also post-translationally modified to a cysteine-sulfinic acid similar to for the native NHase . Although another modification of alphaCys114 could not be identified because of the instability under acidic conditions, the recombinant NHase could be reversibly inactivated by nitric oxide.

Biotechnol Bioeng, 1999 Mar 5, 62(5), 526 - 36
Temperature effects and substrate interactions during the aerobic biotransformation of BTEX mixtures by toluene-enriched consortia and Rhodococcus rhodochrous; Deeb RA et al.; A microbial consortium derived from a gasoline-contaminated aquifer was enriched on toluene (T) in a chemostat at 20 degrees C and was found to degrade benzene (B), ethylbenzene (E), and xylenes (X) . Studies conducted to determine the optimal temperature for microbial activity revealed that cell growth and toluene degradation were maximized at 35 degrees C . A consortium enriched at 35 degrees C exhibited increased degradation rates of benzene, toluene, ethylbenzene, and xylenes in single-substrate experiments; in BTEX mixtures, enhanced benzene, toluene, and xylene degradation rates were observed, but ethylbenzene degradation rates decreased . Substrate degradation patterns over a range of BTEX concentrations (0 to 80 mg/L) for individual aromatics were found to differ significantly from patterns for aromatics in mixtures . Individually, toluene was degraded fastest, followed by benzene, ethylbenzene, and the xylenes . In BTEX mixtures, degradation followed the order of ethylbenzene, toluene, and benzene, with the xylenes degraded last . A pure culture isolated from the 35 degrees C-enriched consortium was identified as Rhodococcus rhodochrous . This culture was shown to degrade each of the BTEX compounds, individually and in mixtures, following the same degradation patterns as the mixed cultures . Additionally, R . rhodochrous was shown to utilize benzene, toluene, and ethylbenzene as primary carbon and energy sources . Studies conducted with the 35 degrees C-enriched consortium and R . rhodochrous to evaluate potential substrate interactions caused by the concurrent presence of multiple BTEX compounds revealed a range of substrate interaction patterns including no interaction, stimulation, competitive inhibition, noncompetitive inhibition, and cometabolism . In the case of the consortium, benzene and toluene degradation rates were slightly enhanced by the presence of o-xylene, whereas the presence of toluene, benzene, or ethylbenzene had a negative effect on xylene degradation rates . Ethylbenzene was shown to be the most potent inhibitor of BTEX degradation by both the mixed and pure cultures . Attempted quantification of these inhibition effects in the case of the consortium suggested a mixture of competitive and noncompetitive inhibition kinetics . Benzene, toluene, and the xylenes had a negligible effect on the biodegradation of ethylbenzene by both cultures . Cometabolism of o-, m-, and p-xylene was shown to be a positive substrate interaction .

Osaka City Med J, 1998 Dec, 44(2), 201 - 17
Granuloma formation and in vitro macrophage activation in mice by mycoloyl glycolipids from Nocardia asteroides and related taxa; Han Y et al.; Cord factor (trehalose 6,6'-dimycolate: TDM) is a well-known toxic glycolipid in Mycobacterium tuberculosis . We isolated various mycoloyl glycolipids from Nocardia asteroides 23,167 and related species which are closely related taxonomically to Mycobacterium . Since Nocardia is also an opportunistic pathogen co-infected with HIV, we examined in vivo granuloma formation and the in vitro macrophage activation in mice . We found that cord factor (TDM) and glucose monomycolate (GM) from Nocardia asteroides and Rhodococcus species with shorter chain mycolic acids also exhibited distinctive granuloma-forming activity in lungs, spleen and liver in mice and in vitro induction of prostaglandin E2 (PGE2) and interleukin 1 (IL-1) synthesis . We also found that mycoloyl glycolipids possessing trehalose or glucose as a carbohydrate moiety exhibited immunomodulatory activity, and that mycoloyl glycolipids with longer chain mycolic acids exhibited stronger activity in mice than did those with shorter chain mycolic acids . The mycoloyl glycolipids from Nocardia asteroides directly activated macrophages . Stimulation of above concentration with 0.16 microgram/ml of TDM or 0.8 microgram/ml of GM markedly enhanced production of PGE2 by mouse peritoneal macrophages . At higher concentrations above 100 micrograms/ml of TDM or 500 micrograms/ml of GM, IL-1 release was also enhanced.

Eur J Biochem, 1999 Mar, 260(2), 446 - 52
Heterologous expression of alkene monooxygenase from Rhodococcus rhodochrous B-276; Smith TJ et al.; 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.

J Bacteriol, 1999 Apr, 181(7), 2094 - 101
Purification of a glutathione S-transferase and a glutathione conjugate-specific dehydrogenase involved in isoprene metabolism in Rhodococcus sp . strain AD45; van Hylckama Vlieg JE et al.; A glutathione S-transferase (GST) with activity toward 1, 2-epoxy-2-methyl-3-butene (isoprene monoxide) and cis-1, 2-dichloroepoxyethane was purified from the isoprene-utilizing bacterium Rhodococcus sp . strain AD45 . The homodimeric enzyme (two subunits of 27 kDa each) catalyzed the glutathione (GSH)-dependent ring opening of various epoxides . At 5 mM GSH, the enzyme followed Michaelis-Menten kinetics for isoprene monoxide and cis-1, 2-dichloroepoxyethane, with Vmax values of 66 and 2.4 micromol min-1 mg of protein-1 and Km values of 0.3 and 0.1 mM for isoprene monoxide and cis-1,2-dichloroepoxyethane, respectively . Activities increased linearly with the GSH concentration up to 25 mM . 1H nuclear magnetic resonance spectroscopy showed that the product of GSH conjugation to isoprene monoxide was 1-hydroxy-2-glutathionyl-2-methyl-3-butene (HGMB) . Thus, nucleophilic attack of GSH occurred on the tertiary carbon atom of the epoxide ring . HGMB was further converted by an NAD+-dependent dehydrogenase, and this enzyme was also purified from isoprene-grown cells . The homodimeric enzyme (two subunits of 25 kDa each) showed a high activity for HGMB, whereas simple primary and secondary alcohols were not oxidized . The enzyme catalyzed the sequential oxidation of the alcohol function to the corresponding aldehyde and carboxylic acid and followed Michaelis-Menten kinetics with respect to NAD+ and HGMB . The results suggest that the initial steps in isoprene metabolism are a monooxygenase-catalyzed conversion to isoprene monoxide, a GST-catalyzed conjugation to HGMB, and a dehydrogenase-catalyzed two-step oxidation to 2-glutathionyl-2-methyl-3-butenoic acid.

Chest, 1999 Mar, 115(3), 889 - 92
Pulmonary malacoplakia associated with Rhodococcus equi infection in a patient with AIDS; Shin MS et al.; An AIDS patient with a cavitary lung lesion was found to have pulmonary malacoplakia associated with Rhodococcus equi infection . The diagnosis was based on the typical histologic features of transbronchial biopsy and a positive bacterial culture . All 13 reported cases of AIDS patients with pulmonary malacoplakia were associated with R equi . The recognition of this unique entity is important because of its responsiveness to therapy.

Biochem Biophys Res Commun, 1999 Mar 16, 256(2), 415 - 8
Hydrazide synthesis: novel substrate specificity of amidase; Kobayashi M et al.; The amidase from Rhodococcus rhodochrous J1, which hydrolyses amide to acid and ammonia, was found to catalyze the synthesis of hydrazide using hydrazine as a substrate . This is the first report on the hydrazide synthesis through enzymatic reactions . The enzyme also acted on benzoic acid in the presence of hydrazine, yielding benzoic hydrazide . Together with the finding that benzoic hydrazide was converted into benzoic acid (when it was used as a substrate in the absence of hydrazine), these unique characteristics suggest that the reaction route for the formation of the acid from the hydrazide and that of the hydrazide from the acid are reversible to each other via the acyl-enzyme . Not only aromatic hydrazides but also aliphatic hydrazides were synthesized from the corresponding amides and hydrazine .

Antonie Van Leeuwenhoek, 1998 Jul-Oct, 74(1-3), 175 - 88
Molecular characterisation of a Rhodococcus ohp operon; Powell JA et al.; The ohp operon of Rhodococcus strain V49 consists of five genes, ohpR, ohpA, ohpB, ohpC and ohpD which encode putative regulator and transport proteins and confirmed monooxygenase, hydroxymuconic semialdehyde hydrolase and catechol 2,3-dioxygenase enzymes, respectively . These enzymes catalyse the conversion of 3-(2-hydroxyphenyl)propionic acid to the corresponding linear product via a meta-cleavage pathway . Confirmation that the ohp gene cluster formed an operon was provided by gene disruption during which expression of Bacillus levansucrase was confirmed in Rhodococcus . Following biochemical assays of cell-free extracts from recombinant Escherichia coli expressing ohpB (monooxygenase), ohpC (hydroxymuconic-semialdehyde hydrolase) and ohpD (catechol 2,3-dioxygenase), the ortho-hydroxyphenylpropionic acid catabolic pathway in Rhodococcus strain V49 (ATCC 19070) has been predicted.

Antonie Van Leeuwenhoek, 1998 Jul-Oct, 74(1-3), 169 - 73
Structural alteration of linear plasmids encoding the genes for polychlorinated biphenyl degradation in Rhodococcus strain RHA1; Fukuda M et al.; Polychlorinated biphenyl (PCB) tolerant derivatives of a strong PCB degrader, Rhodococcus strain RHA1, were selected after growth in the presence of 100 micrograms/ml PCBs . Some of the derivatives did not grow on biphenyl but accumulated a yellow coloured metabolite suggesting a defect in the meta-ring-cleavage compound hydrolase step encoded by the bphD gene . Other derivatives failed to grow on biphenyl and exhibited little PCB transformation activity suggesting a defect in the initial ring-hydroxylation dioxygenase step encoded by the bphA gene . These organisms had a structural alteration in the linear plasmids coding for the bph genes in RHA1, which included the bph gene deletion . When a bphD containing plasmid was introduced into a tolerant derivative, RCD1, which was shown to have bphD deletion, the defect in the growth on biphenyl of RCD1 was overcome . The bph gene deletion seems to play a key role in these tolerant derivatives thereby suggesting that the toxic metabolic intermediate would be a main cause of the growth inhibition of RHA1 in the presence of high concentration PCBs.

Antonie Van Leeuwenhoek, 1998 Jul-Oct, 74(1-3), 155 - 67
Cloning of genes that have environmental and clinical importance from rhodococci and related bacteria; Dabbs ER; Generalised and specialised transduction systems were developed for Rhodococcus by means of bacteriophage Q4 . The latter was used in conjunction with DNA from an unstable genetic element of R . rhodochrous to construct resistance plasmids which replicate in strains of R . equi, R . erythropolis and R . rhodochrous . One of the plasmids, pDA21, was joined with Erythropolis coli suicide vector pEcoR251 to obtain shuttle plasmids maintained in both rhodococci and E . coli . Conjugation between these rhodococcal strains demonstrated all were interfertile with each other and that some of the determinants for this were located on the unstable genetic element . Plasmids derived from this element, such as pDA21, carried the conjugative and self-incompatibility capacities; deletion analysis revealed that DNA necessary for self-incompatibility overlapped with that for arsenic resistance . Rifampicin is one of the principal chemotherapeutic agents used to treat infections by rhodococci and related organisms . The genes responsible for two types of inactivation have been cloned . The sequence of the R . equi DNA responsible for decomposition of the antibiotic strongly resembled those of monooxygenases acting upon phenolic compounds, consistent with the presence of a naphthalenyl moiety in the rifampicin molecule . Antibiotic resistance conferred by the gene was surprisingly specific to the semisynthetic compounds rifampicin (150-fold increase) and rifapentine (70-fold) . Similar specificity was observed with the other inactivation gene cloned, which ribosylates rifampicin at the 23-hydroxyl position . A 60-bp sequence upstream of the monooxygenase and ribosylation genes is strikingly similar suggesting a shared pattern of regulation . Rhodococcal arsenic resistance and azo dye degradation genes have been cloned and characterised.

Antonie Van Leeuwenhoek, 1998 Jul-Oct, 74(1-3), 119 - 32
Desulphurisation of benzothiophene and dibenzothiophene by actinomycete organisms belonging to the genus Rhodococcus, and related taxa; Oldfield C et al.; Desulphurising enzymes remove the sulphur moiety from an organosulphur molecule leaving the carbon skeleton intact . Two kinds of desulphurisation reaction are recognised . The dibenzothiophene (DBT)-specific pathway desulphurises DBT to inorganic sulphite and 2-hydroxybiphenyl (HBP), and the benzothiophene (BTH)-specific pathway desulphurises BTH to 2-(2'-hydroxyphenyl)ethan 1-al (HPEal) and probably inorganic sulphite . The DBT-desulphurisation pathway was originally identified in Rhodococcus erythropolis strain IGTS8 (ATCC 53968), and the BTH-desulphurisation pathway in Gordonia sp . strain 213E (NCIMB 40816) . These organisms do not further metabolise the organic product of desulphurisation . In this article current knowledge of the biochemistry and genetics of the desulphurisation enzymes is reviewed . The need for separate, DBT- and BTH-specific desulphurisation routes is rationalised in terms of the chemical differences between the two compounds . The desulphurisation pathway is compared with other microbial DBT-degrading enzyme systems . Finally some comments are made concerning the application of desulphurisation enzymes for fuel desulphurisation and on the relevance of these enzymes to the ecology of the mycolata (sensu Chun et al, 1996).

Antonie Van Leeuwenhoek, 1998 Jul-Oct, 74(1-3), 107 - 18
Application of whole cell rhodococcal biocatalysts in acrylic polymer manufacture; Hughes J et al.; Rhodococci are ubiquitous in nature and their ability to metabolise a wide range of chemicals, many of which are toxic, has given rise to an increasing number of studies into their diverse use as biocatalysts . Indeed rhodococci have been shown to be especially good at degrading aromatic and aliphatic nitriles and amides and thus they are very useful for waste clean up where these toxic chemicals are present . The use of biocatalysts in the chemical industry has in the main been for the manufacture of high-value fine chemicals, such as pharmaceutical intermediates, though investigations into the use of nitrile hydratase, amidase and nitrilase to convert acrylonitrile into the higher value products acrylamide and acrylic acid have been carried out for a number of years . Acrylamide and acrylic acid are manufactured by chemical processes in vast tonnages annually and they are used to produce polymers for applications such as superabsorbents, dispersants and flocculants . Rhodococci are chosen for use as biocatalysts on an industrial scale for the production of acrylamide and acrylic acid due to their ease of growth to high biomass yields, high specific enzyme activities obtainable, their EFB class 1 status and robustness of the whole cells within chemical reaction systems . Several isolates belonging to the genus Rhodococcus have been shown in our studies to be among the best candidates for acrylic acid preparation from acrylonitrile due to their stability and tolerance to high concentrations of this reactive and disruptive substrate . A critical part of the selection procedure for the best candidates during the screening programme was high purity product with very low residual substrate concentrations, even in the presence of high product concentrations . Additionally the nitrile and amide substrate scavenging ability which enables rhodococci to survive very successfully in the environment leads to the formation of biocatalysts which are suitable for the removal of low concentrations of acrylonitrile and acrylamide in waste streams and for the removal of impurities in manufacturing processes.

Antonie Van Leeuwenhoek, 1998 Jul-Oct, 74(1-3), 99 - 106
Enantioselective biotransformations using rhodococci; Beard TM et al.; The use of enzymes and whole cells in enantioselective biotransformation reactions is briefly reviewed . A Rhodococcus strain is shown to possess nitrile hydratase and amidase activity . The organism can be used for the enantioselective biotransformation of racemic alpha-amino amides to (S) alpha-amino acids with an enantiomeric excess (ee) of > 98% . Enantioselectivity is effectively time independent allowing easy quantitative conversion of racemic mixtures into enantiomerically pure alpha-amino amides and alpha-amino acids . The reaction is effective for a wide range of alpha-substituents . The pH-dependence of the reaction indicates that the alpha-amino amide is bound to the amidase enzyme in its neutral unprotonated form.

Antonie Van Leeuwenhoek, 1998 Jul-Oct, 74(1-3), 89 - 97
Biotransformation of nitriles by rhodococci; Bunch AW; Rhodococci have been shown to be capable of a very wide range of biotransformations . Of these, the conversion of nitriles into amides or carboxylic acids has been studied in great detail because of the biotechnological potential of such activities . Initial investigations used relatively simple aliphatic nitriles . These studies were quickly followed by the examination of the regio- and stereoselective properties of the enzymes involved, which has revealed the potential synthetic utility of rhodococcal nitrile biotransforming enzymes . Physiological studies on rhodococci have shown the importance of growth medium design and bioreactor operation for the maximal conversion of nitriles . This in turn has resulted in some truly remarkable biotransformation activities being obtained, which have been successfully exploited for commercial organic syntheses (e.g . acrylamide production from acrylonitrile) . The two main types of enzyme involved in nitrile biotransformations by rhodococci are nitrile hydratases (amide synthesis) and nitrilases (carboxylic acid synthesis with no amide intermediate released) . It is becoming clear that many rhodococci contain both activities and multiple forms of each enzyme, often induced in a complex way by nitrogen containing molecules . The genes for many nitrile-hydrolysing enzymes have been identified and sequenced . The crystal structure of one nitrile hydratase is now available and has revealed many interesting aspects of the enzyme structure in relationship to its catalytic activity and substrate selectivity.

Antonie Van Leeuwenhoek, 1998 Jul-Oct, 74(1-3), 71 - 82
The putative regulator of catechol catabolism in Rhodococcus opacus 1CP--an IclR-type, not a LysR-type transcriptional regulator; Eulberg D et al.; The catechol catabolic genes catABC from Rhodococcus opacus 1CP have previously been characterized by sequence analysis of the insert cloned on plasmid pRER1 . Now, a 5.1-kb DNA fragment which overlaps with the insert of pRER1 was cloned, yielding pRER2, and subjected to sequencing . Besides three other open reading frames, a gene was detected ca 200 bp upstream of the catechol 1,2-dioxygenase gene catA, which is obviously transcribed divergently from catABC . The protein which can be deduced from this gene, CatR, resembles members of the PobR subfamily of IclR-type regulatory proteins . This finding was unexpected, as all catechol and chlorocatechol gene clusters known thus far from proteobacteria are under control of LysR-type regulators . It was not possible to inactivate catR by homologous recombination . However, heterologously expressed CatR in vitro bound specifically to the intergenic region between catR and catA thereby providing a first indication for a possible involvement of CatR in the regulation of catechol catabolism.

Antonie Van Leeuwenhoek, 1998 Jul-Oct, 74(1-3), 59 - 70
Surface-active lipids in rhodococci; Lang S et al.; Like other hydrocarbon-oxidising bacteria, rhodococci respond to the presence of alkanes by producing biosurfactant molecules to improve their ability to utilise these hydrophobic compounds as growth substrates . In the rhodococci these surfactants are predominantly glycolipids, the majority of which remain cell-bound during unrestricted growth . Most work has been done on the trehalose mycolates formed by Rhodococcus erythropolis, but nitrogen-limited conditions lead to the production of anionic trehalose tetraesters also . As surfactants, these compounds, whether purified or in crude form, are able to reduce the surface tension of water from 72 mN m-1 to a low of 26, thus making them among the most potent biosurfactants known . They are also able to reduce the interfacial tension between water and a hydrophobic phase (e.g . n-hexadecane) from 43 mN m-1 to values less than one (Table 1) . Biosurfactants have about a ten- to 40-fold lower critical micelle concentration than synthetic surfactants . Such properties suggest a range of industrial applications, where a variety of surface-active characteristics are appropriate . Interest in biosurfactants as industrial chemicals results from the toxicity of many petrochemical-derived surfactants . Currently world-wide surfactant production is on a very large scale, and the demand for them is increasing . However, the drive towards less environmentally damaging chemicals makes biosurfactants attractive as they have lower toxicity . The reason they have not achieved a significant market share is the cost of production, which is considerably higher than for synthetic surfactants . This problem is being addressed using several strategies . An approach where there is great scope for improvement with the rhodococci is an understanding of the genetic basis of glycolipid production, which is largely unknown . They may find applications in the near future in the environmental remediation industries, where the requirement for purified molecules is of less importance . This review summarises knowledge of the chemistry, biochemistry and production of Rhodococcus surface-active lipids . Where they have been used, or there is potential for use, in industrial applications is discussed.

Antonie Van Leeuwenhoek, 1998 Jul-Oct, 74(1-3), 21 - 5
Diversity of isolates of Rhodococcus equi from Australian thoroughbred horse farms; Morton AC et al.; Pulsed field gel electrophoresis of restriction endonuclease digested genomic DNA from a collection of clinical isolates of Rhodococcus equi was used to compare strain diversity on different Thoroughbred horse farms over time . Restricted diversity was found among the isolates tested, as the same strains were detected on multiple farms and in multiple years . Marked variation occurred in strain prevalence with some strains being represented by single isolates, and the most prevalent by 26 isolates . There were dominant strains on some farms and the prevalence of some strains differed between farms . Infection with multiple strains was noted in some cases where multiple isolates from a single foal were examined.

Antonie Van Leeuwenhoek, 1998 Jul-Oct, 74(1-3), 3 - 20
Rhodococcal systematics: problems and developments; Goodfellow M et al.; Various approaches that have been used in the development of a system of classification for the genus Rhodococcus are discussed . The application of chemotaxonomic, molecular systematic and numerical phenetic methods have greatly contributed to improvements in the systematics of rhodococci and related mycolic-acid containing actinomycetes . The genus currently encompasses twelve validly described species but improved diagnostic methods are needed to distinguish between them . In addition, evidence from 16S ribosomal RNA sequencing suggests that the genus is still heterogeneous.

J Struct Biol, 1998 Dec 15, 124(2-3), 179 - 88
Late events in the assembly of 20S proteasomes; Mayr J et al.; Electron microscopy and STEM mass measurements have been used to characterize late intermediates in the assembly pathway of wildtype and mutant Rhodococcus proteasomes . A proteolytically inactive and processing-incompetent mutant, betaK33A, allowed a short-lived late intermediate of the pathway to be captured, the preholoproteasome . In this fully assembled 20S complex the 14 propeptides with an aggregate mass of 100 kDa fill the whole central cavity and most of the two antechambers . It is further shown that in wildtype Rhodococcus proteasomes the propeptides are degraded in a processive manner undergoing multiple cleavages before the products are discharged and the inner cavities are cleared . It appears that the docking of two half-proteasomes, i.e., preholoproteasome formation, is sufficient to trigger autocleavage of the Gly-1/Thr1 bond necessary for active site formation and the subsequent degradation of the propeptides .

Biochemistry, 1999 Feb 23, 38(8), 2326 - 39
Phenylalanine dehydrogenase from Rhodococcus sp . M4: high-resolution X-ray analyses of inhibitory ternary complexes reveal key features in the oxidative deamination mechanism; Vanhooke JL et al.; The molecular structures of recombinant L-phenylalanine dehydrogenase from Rhodococcus sp . M4 in two different inhibitory ternary complexes have been determined by X-ray crystallographic analyses to high resolution . Both structures show that L-phenylalanine dehydrogenase is a homodimeric enzyme with each monomer composed of distinct globular N- and C-terminal domains separated by a deep cleft containing the active site . The N-terminal domain binds the amino acid substrate and contributes to the interactions at the subunit:subunit interface . The C-terminal domain contains a typical Rossmann fold and orients the dinucleotide . The dimer has overall dimensions of approximately 82 A x 75 A x 75 A, with roughly 50 A separating the two active sites . The structures described here, namely the enzyme.NAD+.phenylpyruvate, and enzyme . NAD+.beta-phenylpropionate species, represent the first models for any amino acid dehydrogenase in a ternary complex . By analysis of the active-site interactions in these models, along with the currently available kinetic data, a detailed chemical mechanism has been proposed . This mechanism differs from those proposed to date in that it accounts for the inability of the amino acid dehydrogenases, in general, to function as hydroxy acid dehydrogenases.

Curr Opin Chem Biol, 1999 Feb, 3(1), 16 - 21
Stereoselectivities of microbial epoxide hydrolases; Orru RV et al.; Epoxide hydrolases from bacterial and fungal sources are highly versatile biocatalysts for the asymmetric hydrolysis of epoxides on a preparative scale . Besides kinetic resolution, which yields the corresponding enantiomerically enriched vicinal diol and the remaining nonconverted epoxide, enantioconvergent processes are also possible, which lead to the formation of a single enantiomeric diol from a racemic oxirane . The data available to date indicate that the enantioselectivities of enzymes from certain microbial sources can be correlated to the substitutional pattern of various types of substrates: red yeasts (e.g . Rhodotorula or Rhodosporidium sp.) give best enantioselectivities with monosubstituted oxiranes; fungal cells (e.g . from Aspergillus and Beauveria sp.) are best suited for styrene oxide-type substrates; bacterial enzymes, on the other hand (in particular from Actinomycetes such as Rhodococcus and Nocardia sp.) are the biocatalysts of choice for more highly substituted 2,2- and 2,3-disubstituted epoxides.

J Bacteriol, 1999 Feb, 181(4), 1189 - 95
Hydride-Meisenheimer complex formation and protonation as key reactions of 2,4,6-trinitrophenol biodegradation by Rhodococcus erythropolis; Rieger PG et al.; Biodegradation of 2,4,6-trinitrophenol (picric acid) by Rhodococcus erythropolis HLPM-1 proceeds via initial hydrogenation of the aromatic ring system . Here we present evidence for the formation of a hydride-Meisenheimer complex (anionic sigma-complex) of picric acid and its protonated form under physiological conditions . These complexes are key intermediates of denitration and productive microbial degradation of picric acid . For comparative spectroscopic identification of the hydride complex, it was necessary to synthesize this complex for the first time . Spectroscopic data revealed the initial addition of a hydride ion at position 3 of picric acid . This hydride complex readily picks up a proton at position 2, thus forming a reactive species for the elimination of nitrite . Cell extracts of R . erythropolis HLPM-1 transform the chemically synthesized hydride complex into 2,4-dinitrophenol . Picric acid is used as the sole carbon, nitrogen, and energy source by R . erythropolis HLPM-1.

Adv Biochem Eng Biotechnol, 1999, 63, 145 - 67
Epoxide hydrolases and their synthetic applications; Orru RV et al.; Chiral epoxides and 1,2-diols, which are central building blocks for the asymmetric synthesis of bioactive compounds, can be obtained by using enzymes--i.e . epoxide hydrolases--which catalyse the enantioselective hydrolysis of epoxides . These biocatalysis have recently been found to be more widely distributed in fungi and bacteria than previously expected . Sufficient sources from bacteria, such as Rhodococcus and Nocardia spp., or fungi, as for instance Aspergillus and Beauveria spp., have now been identified . The reaction proceeds via an SN2-specific opening of the epoxide, leading to the formation of the corresponding trans-configured 1,2-diol . For the resolution of racemic monosubstituted and 2,2- or 2,3-disubstituted substrates, various fungi and bacteria have been shown to possess excellent enantioselectivities . Additionally, different methods, which lead to the formation of the optically pure product diol in a chemical yield far beyond the 50% mark (which is intrinsic to classic kinetic resolutions), are discussed . In addition, the use of non-natural nucleophiles such as azides or amines provides access to enantiomerically enriched vicinal azido- and amino-alcohols . The synthetic potential of these enzymes for asymmetric synthesis is illustrated with recent examples, describing the preparation of some biologically active molecules.

Appl Environ Microbiol, 1999 Feb, 65(2), 853 - 5
Effect of aromatic compounds on cellular fatty acid composition of rhodococcus opacus
Tsitko IV IV, Zaitsev GM, Lobanok AG, Salkinoja-Salonen MS.
In cells of Rhodococcus opacus GM-14, GM-29, and 1CP, the contents of branched (10-methyl) fatty acids increased from 3% to 15 to 34% of the total fatty acids when the cells were grown on benzene, phenol, 4-chlorophenol, chlorobenzene, or toluene as the sole source of carbon and energy, in comparison with cells grown on fructose . In addition, the content of trans-hexadecenoic acid increased from 5% to 8 to 18% with phenol or chlorophenol as the carbon source . The 10-methyl branched fatty acid content of R . opacus GM-14 cells increased in a dose-related manner following exposure to phenol or toluene when toluene was not utilized as the growth substrate . The results suggest that 10-methyl branched fatty acids may participate in the adaptation of R . opacus to lipophilic aromatic compounds.

Biochem Biophys Res Commun, 1998 Dec 30, 253(3), 662 - 6
Nitrilase catalyzes amide hydrolysis as well as nitrile hydrolysis; Kobayashi M et al.; While amides were reported to be completely inert as substrates for all nitrilases reported to date, the nitrilase from Rhodococcus rhodochrous J1, which catalyzes the hydrolytic cleavage of the C-N triple bond in nitrile to form acid and ammonium, was surprisingly found to catalyze hydrolysis of amide to acid and ammonium stoichiometrically . This nitrilase exhibited a Km of 2.94 mM for benzamide, similar to that for benzonitrile as the original substrate (2.10 mM), but the Vmax for benzamide was six orders of magnitude lower than that for benzonitrile . Benzamide inhibited the nitrilase reaction in a reversible, apparently competitive manner . A mutant nitrilase containing alanine or serine instead of Cys165, which is essential for nitrilase catalytic activity, showed no amidase activity . This observation demonstrated that Cys165 plays a crucial role in the hydrolysis of amides as well as nitriles . Together with some reports that certain nitrilases were previously noted to produce low amounts of amide as a by-product from nitrile, the above unexpected findings suggested the existence of a common tetrahedral intermediate in the nitrilase reaction involving nitrile or amide as a substrate.

Nippon Rinsho, 1998 Dec, 56(12), 3008 - 16
{Biochemistry and bioactivities of mycobacterial components}; Yano I; The most characteristic pathological change in mycobacterial infection is caseous necrosis followed by tuberculous cavity formation due to the cellular immunity induced by antigenic proteins and adjuvant active cell wall components . Mycobacterial cell well contains unique hydrophobic compounds possessing mycolic acids (a long branched-chain high molecular weight fatty acid) and shows distinctive properties such as acid-fastness and wax-like hydrophobicity . Mycobacteria do not produce exotoxin and the virulence cannot be determined by a single toxic substance, but the cell wall components to contact with the host cells are the most important surface molecule at the early stage of infection . Cord factor (trehalose 6,6'-dimycolate) is the most classical virulence factor which is lethally toxic for mice . However, cord factor exists among various species of mycobacteria and even in Nocardia and Rhodococcus . Furthermore, cord factor can produce granulomas without protein antigen in mice and it shows antitumor or non-specific prevention promotion of infection and induction promotion of various cytokines . Sulfolipid (tetracyl trehalose sulfate) also plays a role as a virulence factor by phagocytic process inhibition . Glycopeptidolipid (GPL) from M . avium and phenolglycolipid (PGL) from M . leprae also appeared to be immunomodulatory molecules which inhibit the developing of cellular immunity . Lipoarabinomannan (LAM) is also unique amphipatic molecule, like gram-negative endotoxin . Here, we discuss the involvement of various surface molecutes to contribute to pathogenesis in mycobacterial infection and immunity.

Microbiol Res, 1998 Nov, 153(3), 239 - 45
Screening of xenobiotic compounds degrading microorganisms using biosensor techniques; Beyersdorf-Radeck B et al.; A screening device based on microorganisms immobilised onto a Clark-type oxygen electrode was used to monitor the potential of these microorganisms for the degradation and detection of xenobiotic compounds especially their chlorinated derivatives . The sensitivity and specificity of various species of Pseudomonas, Sphinomonas, Ralstonia, Rhodococcus were characterised in relation to xenobiotic compounds by using biosensor techniques . The following groups of xenobiotics were subjects of investigation: chlorophenols, chlorobenzoates, 2,4-D, PCB, dibenzofurane and their putative intermediates . Using this simple setup it proved possible to screen microbial strains for their potential to catabolize aromatic and chloroaromatic compounds under oxygen consumption . In a kinetic regime, a reproducible signal was obtained within minutes . Based on these results the sensor technique was a suitable method for the rapid characterization of microorganisms and allowed to gather information about the substrate spectrum.

FEMS Immunol Med Microbiol, 1998 Dec, 22(4), 329 - 33
Cutaneous malakoplakia in pigs inoculated with Rhodococcus equi; Madarame H et al.; Cutaneous malakoplakia was observed in pigs inoculated intramuscularly with Rhodococcus equi strains of intermediate virulence . Macroscopically, the inoculation sites showed the indurated swelling of the skin . Histopathologically, abscess formation with histiocytic granulomatous reaction was observed . Many macrophages contained target or owl-eye shaped hematoxyphil intracytoplasmic inclusions or calcosherites (Michaelis-Gutmann bodies) of various sizes . The Michaelis-Gutmann bodies were also seen outside of the macrophages . Histochemically, most Michaelis-Gutmann bodies stained positively with the von Kossa silver method and periodic acid Schiff . Immunohistochemically, some of Michaelis-Gutmann bodies were stained by two rabbit polyclonal antibodies (rabbit anti-A5 serum and rabbit anti-ATCC 33701 serum) and a mouse monoclonal antibody (anti-20-kDa antigen monoclonal antibody) . This is the first report of cutaneous malakoplakia in domestic animals, which also revealed the relationship between R . equi infection and malakoplakia immunohistochemically . This experimental swine model is useful to investigate the morphogenesis of Michaelis-Gutmann bodies in malakoplakia through chronological skin biopsies.

Appl Environ Microbiol, 1999 Jan, 65(1), 181 - 8
Microbial desulfurization of a crude oil middle-distillate fraction: analysis of the extent of sulfur removal and the effect of removal on remaining sulfur; Grossman MJ et al.; Rhodococcus sp . strain ECRD-1 was evaluated for its ability to desulfurize a 232 to 343 degrees C middle-distillate (diesel range) fraction of Oregon basin (OB) crude oil . OB oil was provided as the sole source of sulfur in batch cultures, and the extent of desulfurization and the chemical fate of the residual sulfur in the oil after treatment were determined . Gas chromatography (GC), flame ionization detection, and GC sulfur chemiluminesce detection analysis were used to qualitatively evaluate the effect of Rhodococcus sp . strain ECRD-1 treatment on the hydrocarbon and sulfur content of the oil, respectively . Total sulfur was determined by combustion of samples and measurement of released sulfur dioxide by infrared absorption . Up to 30% of the total sulfur in the middle distillate cut was removed, and compounds across the entire boiling range of the oil were affected . Sulfur K-edge X-ray absorption-edge spectroscopy was used to examine the chemical state of the sulfur remaining in the treated OB oil . Approximately equal amounts of thiophenic and sulfidic sulfur compounds were removed by ECRD-1 treatment, and over 50% of the sulfur remaining after treatment was in an oxidized form . The presence of partially oxidized sulfur compounds indicates that these compounds were en route to desulfurization . Overall, more than two-thirds of the sulfur had been removed or oxidized by the microbial treatment.

J Basic Microbiol, 1998, 38(4), 257 - 267
Etherolytic cleavage of 4-(2,4-dichlorophenoxy)butyric acid and 4-(4-chloro-2-methylphenoxy)butyric acid by species of Rhodococcus and Aureobacterium isolated from an alkaline environment; Mertingk H et al.; Bacterial strains were isolated from the concrete rubble of a demolished herbicide production plant . The predominant feature of these strains was the etherolytic cleavage of 4-(2,4-dichlorophenoxy)butyric acid (DCPB)1) and 4-(4-chloro-2-methylphenoxy)butyric acid (MCPB) while liberating 2,4-dichlorophenol (DCP) and 4-chloro-2-methylphenol (MCP) respectively . Some of the isolates were identified by 16S rDNA sequence analysis and shown to belong to the genera Aureobacterium sp . (strain K2-17) and Rhodococcus (Rh . erythropolis K2-12) . The other strains isolated clustered into these two groups according to fatty acid analysis . Etherolytic cleavage proceeded under neutral to alkaline conditions with an optimum at around pH 8.5 . With Aureobacterium sp . No . K2-17, the degradation rate was zero at a pH of 6 but as much as 60% of the maximum activity was observed at pH 10.5 . With Rh . erythropolis K2-12, by contrast, pronounced activity was detected at pH 6.5 while degradation was no longer observed at pH 10.5 . The maximum rates of cleavage were about 1 mmol DCPB/h.g dry mass with Aureobacterium sp . No . K2-17 and about 0.6 mmol DCPB/h.g dry mass with Rh . erythropolis K2-12 . DCPB and MCPB were utilized to the same extent . Substrate cleavage and product formation (DCP) proceeded at almost equal rates with Aureobacterium sp . No . K2-17 and Rh . erythropolis K2-12, which indicates that this compound was not further metabolized . Only phenoxybutyric acid compounds served as substrates; phenoxyacetic acid and phenoxypropionic acid derivatives were not utilized by these strains.

Eur J Clin Microbiol Infect Dis, 1998 Oct, 17(10), 737 - 9
Rhodococcus equi and Nocardia brasiliensis infection of the brain and liver in a patient with acute nonlymphoblastic leukemia; Akan H et al.; Resolution of neutropenia is usually followed by resolution of fever in patients with febrile neutropenia . However, in some cases fever continues even when the patient is no longer neutropenic . Described here is a case of acute myeloblastic leukemia complicated by brain abscess, pulmonary nodules, and hepatic lesions . The patient's fever had continued after the neutropenia resolved; brain and hepatic cultures grew Rhodococcus equi and Nocardia brasiliensis . Although Rhodococcus infections occur frequently in patients with HIV infection, they are uncommon in patients with acute leukemia.

J Bacteriol, 1999 Jan, 181(1), 149 - 52
3-nitroadipate, a metabolic intermediate for mineralization of 2, 4-dinitrophenol by a new strain of a Rhodococcus species; Blasco R et al.; The bacterial strain RB1 has been isolated by enrichment cultivation with 2,4-dinitrophenol as the sole nitrogen, carbon, and energy source and characterized, on the basis of 16S rRNA gene sequence comparison, as a Rhodococcus species closely related to Rhodococcus opacus . Rhodococcus sp . strain RB1 degrades 2,4-dinitrophenol, releasing the two nitro groups from the compound as nitrite . The release of nitro groups from 2,4-dinitrophenol occurs in two steps . First, the 2-nitro group is removed as nitrite, with the production of an aliphatic nitro compound identified by 1H nuclear magnetic resonance and mass spectrometry as 3-nitroadipate . Then, this metabolic derivative is further metabolized, releasing its nitro group as nitrite . Full nitrite assimilation upon reduction to ammonia requires that an additional carbon source be supplied to the medium.

J Clin Microbiol, 1999 Jan, 37(1), 99 - 102
Rapid identification of clinically relevant Nocardia species to genus level by 16S rRNA gene PCR; Laurent FJ et al.; Two regions of the gene coding for 16S rRNA in Nocardia species were selected as genus-specific primer sequences for a PCR assay . The PCR protocol was tested with 60 strains of clinically relevant Nocardia isolates and type strains . It gave positive results for all strains tested . Conversely, the PCR assay was negative for all tested species belonging to the most closely related genera, including Dietzia, Gordona, Mycobacterium, Rhodococcus, Streptomyces, and Tsukamurella . Besides, unlike the latter group of isolates, all Nocardia strains exhibited one MlnI recognition site but no SacI restriction site . This assay offers a specific and rapid alternative to chemotaxonomic methods for the identification of Nocardia spp . isolated from pathogenic samples.

J Bacteriol, 1998 Dec, 180(24), 6668 - 73
The modified beta-ketoadipate pathway in Rhodococcus rhodochrous N75: enzymology of 3-methylmuconolactone metabolism; Cha CJ et al.; Rhodococcus rhodochrous N75 is able to metabolize 4-methylcatechol via a modified beta-ketoadipate pathway . This organism has been shown to activate 3-methylmuconolactone by the addition of coenzyme A (CoA) prior to hydrolysis of the butenolide ring . A lactone-CoA synthetase is induced by growth of R . rhodochrous N75 on p-toluate as a sole source of carbon . The enzyme has been purified 221-fold by ammonium sulfate fractionation, hydrophobic chromatography, gel filtration, and anion-exchange chromatography . The enzyme, termed 3-methylmuconolactone-CoA synthetase, has a pH optimum of 8.0, a native Mr of 128,000, and a subunit Mr of 62,000, suggesting that the enzyme is homodimeric . The enzyme is very specific for its 3-methylmuconolactone substrate and displays little or no activity with other monoene and diene lactone analogues . Equimolar amounts of these lactone analogues brought about less than 30% (most brought about less than 15%) inhibition of the CoA synthetase reaction with its natural substrate.

FEBS Lett, 1998 Nov 20, 439(3), 325 - 8
The catalytic mechanism of amidase also involves nitrile hydrolysis; Kobayashi M et al.; The amidase from Rhodococcus rhodochrous J1, which hydrolyzes an amide to an acid and ammonium, was surprisingly found to catalyze the hydrolytic cleavage of the C-N triple bond in a nitrile to form an acid and ammonium stoichiometrically . The amidase exhibited a Km of 3.26 mM for benzonitrile in contrast to that of 0.15 mM for benzamide as the original substrate, but the Vmax for benzonitrile was about 116000 of that for benzamide . A mutant amidase containing alanine instead of Ser195, which is essential for amidase catalytic activity, showed no nitrilase activity, demonstrating that this residue plays a crucial role in the hydrolysis of nitriles as well as amides.

J Comp Pathol, 1998 Nov, 119(4), 397 - 405
Pathogenicity of Rhodococcus equi strains possessing virulence-associated 15- to 17-kDa and 20-kDa antigens: experimental and natural cases in pigs; Madarame H et al.; The pathogenic role of Rhodococcus equi in pigs remains controversial . Small numbers of pigs were inoculated intravenously (i.v.), or intramuscularly (i.m.) around the mouth, with a virulent, an intermediately virulent, or an avirulent strain of R . equi and killed 14 days later . None showed clinical signs other than transient fever and weight loss . The virulent and intermediately virulent strains were recovered in culture from various organs and lymph nodes of pigs inoculated i.v., but only from the mandibular lymph nodes of pigs inoculated i.m . The avirulent strain was not recovered from any site . None of the pigs developed macroscopically visible lesions, but they showed reactive hyperplasia of the mandibular lymph nodes . The latter contained scattered phagocytic cells, which were labelled immunohistochemically for virulence-associated antigens (15- to 17-kDa antigens or 20-kDa antigen) . Intermediately virulent and virulent strains of R . equi were isolated from mandibular lymph nodes of 5.5% of apparently healthy abattoir pigs (n = 1615) . Virulence-associated antigens were detected in phagocytic cells of culture-positive nodes, but the latter showed no lesions other than reactive lymphoid hyperplasia . The results would seem to question the pathogenic role of R . equi in pigs, and it is speculated that the organism survives in the lymph nodes without causing pathognomonic lesions.

FEBS Lett, 1998 Nov 6, 438(3), 293 - 6
The Rhodococcus erythropolis DCL14 limonene-1,2-epoxide hydrolase gene encodes an enzyme belonging to a novel class of epoxide hydrolases; Barbirato F et al.; Recently, we reported the purification of the novel enzyme limonene-1,2-epoxide hydrolase involved in limonene degradation by Rhodococcus erythropolis DCL14 . The N-terminal amino acid sequence of the purified enzyme was used to design two degenerate primers at the beginning and the end of the 50 amino acids long stretch . Subsequently, the complete limonene-1,2-epoxide hydrolase gene (limA) was isolated from a genomic library of R . erythropolis DCL14 using a combination of PCR and colony hybridization . The limA gene encoded a 149-residue polypeptide with a deduced molecular mass of 16.5 kDa . It was functionally expressed in Escherichia coli . The amino acid sequence of limA contains neither any of the conserved regions of the alpha,beta-hydrolase fold enzymes, to which most of the previously reported epoxide hydrolases belong, nor any of the conserved motifs present in leukotriene A4 hydrolase . The structural data presented in this paper confirm previous physical and biochemical findings {van der Werf et al . (1998) J . Bacteriol . 180, 5052-5057} that limonene-1,2-epoxide hydrolase is the first member of a new class of epoxide hydrolases.

J Med Assoc Thai, 1998 Nov, 81(11), 923 - 6
Uncommon manifestations of opportunistic infections in an HIV infected patient; Chiewchanvit S et al.; A case of an HIV infected patient complicated with Penicillium marneffei and Rhodococcus equi infection is reported . He presented with chronic ulcer as pyoderma gangrenosum-like on his right calf and submandibular lymphadenitis as scrofuloderma-like . Penicillium marneffei and Rhodococcus equi were isolated from the ulcer and lymph node respectively.

Biotechnol Appl Biochem, 1998 Dec, 28 ( Pt 3), 229 - 33
Maximization of cholesterol oxidase production by Rhodococcus equi no . 23 By using response surface methodology; Lee MT et al.; Medium optimization for the production of cholesterol oxidase (EC 1 . 1.3.6) by Rhodococcus equi no . 23 was investigated by using response surface methodology and a central composite design . Results revealed that cholesterol and yeast extract had positive effects and the interaction between any two of three factors had no significant effect on cholesterol oxidase production . The optimized medium was the basal medium with the addition of 2.30 g/l cholesterol, 8.18 g/l yeast extract and 4.10 ml/l Tween 80 . The peak cholesterol oxidase production (0.242 unit/ml) after 60-72 h cultivation was approx . 4-fold that in control medium.

Appl Environ Microbiol, 1998 Nov, 64(11), 4363 - 7
Characterization of the basic replicon of Rhodococcus plasmid pSOX and development of a Rhodococcus-Escherichia coli shuttle vector; Denis-Larose C et al.; The replication region of a 100-kb desulfurization plasmid (pSOX) from Rhodococcus sp . strain X309 was localized to a 4-kb KpnI fragment, and its sequence was determined . The amino acid sequence of one of the predicted open reading frames (ORFs) was related to the putative replication (Rep) protein sequences of the mycobacterial pLR7 family of plasmids . Three of the five predicted ORF products were identified by radiolabelling with the Escherichia coli T7 polymerase/promoter system . In E . coli, the Rep protein of pSOX was apparently synthesized in a shortened form, 21.3 kDa instead of the predicted 41.3 kDa, as a result of an internal initiation . This situation is reminescent of that for some bacterial Rep proteins . A shuttle plasmid was constructed with the pSOX origin, pBluescript II KS-, and the chloramphenicol resistance (Cmr) gene from pRF29 . This new shuttle plasmid was used to demonstrate expression of the Bacillus subtilis sacB gene in a strain of Rhodococcus, rendering it sensitive to the presence of sucrose.

J Biochem (Tokyo), 1998 Nov, 124(5), 1026 - 32
3-Ketosteroid-delta1-dehydrogenase of Rhodococcus rhodochrous: sequencing of the genomic DNA and hyperexpression, purification, and characterization of the recombinant enzyme; Morii S et al.; The gene encoding 3-ketosteroid-Delta1-dehydrogenase from Rhodococcus rhodochrous was cloned and sequenced . The gene (ksdD) consists of 1,536 nucleotides and encodes an enzyme protein of 511 amino acid residues . The amino terminal methionine residue was deleted in the mature protein . The amino acids involved in the flavin binding site are conserved in the dehydrogenase sequence . The deduced amino acid sequence is highly homologous to that from Arthrobacter simplex but less so to that from Pseudomonas testosteroni . Upstream of the gene was located a heat shock protein gene, dnaJ, and downstream, a gene of a hypothetical protein . The enzyme gene was ligated with an expression vector to construct a plasmid pDEX-3 and introduced into Escherichia coli cells . The transformed cells hyperexpressed the 3-ketosteroid-Delta1-dehydrogenase as an active and soluble protein at more than 30 times the level of R . rhodochrous cells . Purification of the recombinant 3-ketosteroid-Delta1-dehydrogenase from the E . coli cells by a simplified procedure yielded about 13 mg of enzyme protein/liter of the bacterial culture . The purified recombinant dehydrogenase exhibited identical molecular and catalytic properties to the R . rhodochrous enzyme.

J Basic Microbiol, 1998, 38(4), 241 - 55
Utilization of quinate and p-hydroxybenzoate by actinomycetes: key enzymes and taxonomic relevance; Grund E et al.; 474 strains of the actinomycete genera Streptomyces (including species of the former genera Chainia and Streptoverticillium), Pseudonocardia and Micromonospora were examined for their ability to degrade quinate (Q) and p-hydroxybenzoate (pHB); selected strains were also tested for their capacity to catabolize benzoate (B) . Whereas in the case of Q (5-10 g/l of a mineral salts agar medium) the growth response signalizes assimilation, pHB has to be supplied in lower concentration (routinely 0.3 g/l together with small amounts of peptone and yeast extract in liquid broth), and its degradation has to be determined spectrophotometrically . 27% of the streptomycete strains were able to grow with Q, and 57% with pHB . The three strains of "Chainia" that were tested metabolized Q and pHB, but none of the fourty species of "Streptoverticillium" showed this ability . 80% of the 30 strains of Psn . autotrophica grew with Q, and 100% degraded pHB and B . Two of the five Micromonospora strains gave a positive response with pHB, but not with Q.-Toluene treated cells (preincubated with Q, pHB or B, respectively) gave a positive Rothera reaction with protocatechuate or catechol respectively, thus demonstrating that these organisms employed the beta-ketoadipate pathway (orthofission) for the degradation of Q, pHB and B . The assay of five relevant enzymes in cell-free extracts of nine selected organisms showed that in nocardioform actinomycetes (Pseudonocardia, Rhodococcus) all enzymes of the protocatechuate branch of the ketoadipate pathway seem to be induced by beta-ketoadipate as demonstrated here for protocatechuate-3,4-dioxygenase . In contrast, in Streptomyces this enzyme appears to be induced by its substrate, protocatechuate, whereas the regulation of the other enzymes of this pathway remains to be elucidated.

Extremophiles, 1998 Aug, 2(3), 269 - 77
Taxonomy and biotransformation activities of some deep-sea actinomycetes; Colquhoun JA et al.; Deep-sea soft sediments from trench systems and depths in the northwestern Pacific Ocean ranging from less than 300 to 10,897 m in depth have been analyzed for three target genera of actinomycetes: Micromonospora, Rhodococcus, and Streptomyces . Only culturable strains, recovered at atmospheric pressure on selective isolation media, have been examined to date . Maximum recoveries of culturable bacteria were greater that 10(7)/ml wet g sediment, but actinomycetes comprised a small proportion of this population (usually less than 1%) . The target actinomycetes were isolated at all depths except from the Mariana Trench sediments . Actinomycete colonies were defined initially on the basis of colony morphologies, and preliminary identification then was made by chemotaxonomic tests . Pyrolysis mass spectrometry (PyMS) of deep-sea mycolic acid-containing actinomycetes gave excellent correspondence with numerical (phenetic) taxonomic analyses and subsequently was adopted as a rapid procedure for assessing taxonomic diversity . PyMS analysis enabled several clusters of deep-sea rhodococci to be distinguished that are quite distinct from all type strains . 16S rRNA gene sequence analysis has revealed that several of these marine rhodococci have sequences that are very similar to certain terrestrial species of Rhodococcus and to Dietzia . There is evidence for the intrusion of terrestrial runoff into these deep trench systems, and the inconsistency of the phenotypic and molecular taxonomies may reflect recent speciatiion events in actinomycetes under the high-pressure conditions of the deep sea . The results of DNA-DNA pairing experiments point to the novelty of Rhodococcus strains recovered from hadal depths in the Izu Bonin Trench . Biotransformation studies of deep-sea bacteria have focused on nitrile compounds . Nitrile-metabolizing bacteria, closely related to rhodococci, have been isolated that grow well at low temperature, high salt concentrations, and high pressures, suggesting that they are of marine origin or have adapted to the deep-sea environment.

Microbiology, 1998 Sep, 144 ( Pt 9), 2545 - 53
Isolation of a unique benzothiophene-desulphurizing bacterium, Gordona sp . strain 213E (NCIMB 40816), and characterization of the desulphurization pathway; Gilbert SC et al.; Gordona sp . strain 213E (NCIMB 40816) grew in pure culture in a mineral salts medium containing fructose as a source of carbon and energy, and benzothiophene (BTH) as the sole source of sulphur . During growth a phenolic compound accumulated, as indicated by the production of a blue colour on addition of Gibb's reagent . Therefore this pathway is analogous to the dibenzothiophene (DBT) desulphurization pathway of Rhodococcus sp . strain IGTS8, in which 2-hydroxybiphenyl accumulates during growth with DBT as the sole sulphur source . Ethyl acetate extraction of the culture medium yielded the metabolites benzothiophene s-oxide (BTHO), benzothiophene s,s-dioxide (BTHO2), benzo{c}{1,2}oxathiin 6-oxide (BcOTO), 2-(2'-hydroxyphenyl) ethan 1-al (HPEal) and benzofuran (BFU) . The deduced pathway for BTH desulphurization is BTH-->BTHO-->BTHO2-->HPESi(-)-->HPEal . HPESi- is (Z)-2-(2'-hydroxyphenyl)ethen 1-sulphinate, the stable aqueous-solution form of BcOTO . It was concluded that HPEal was the Gibb's-reagent-reactive phenolic compound which accumulated in the culture medium of strain 213E during growth, and that the presence of BFU was due to partial condensation of HPEal during the ethyl acetate extraction procedure . Gordona sp . strain 213E was unable to grow in a mineral salts medium containing fructose as a source of carbon and energy and DBT as the sole sulphur source . BTH-desulphurization-active cells (grown using BTH as sole sulphur source) were unable to desulphurize DBT . Likewise Rhodococcus sp . strain IGTS8 was unable to grow using BTH as the sole sulphur source, and DBT-desulphurization-active cells of strain IGTS8 (grown using DBT as sole sulphur source) were unable to desulphurize BTH . This absence of cross-reactivity is discussed in terms of fundamental differences in the chemistry of the DBT- and BTH-desulphurization reactions.

Res Microbiol, 1997 Dec, 148(9), 799 - 809
Identification of Rhodococcus, Gordona and Dietzia species using carbon source utilization tests ("Biotype-100" strips); Bizet C et al.; The "Biotype-100" identification system (BioMerieux, La Balme-Ies-Grottes, France) based on carbon source utilization was evaluated for its ability to discriminate among 10 species of Rhodococcus, 7 species of Gordona and one species of Dietzia . The type strains of three species of Tsukamurella and 8 species of Nocardia were also included in the study . Results were compared with chemotaxonomic and conventional data . Carbon source utilization was shown to be reliable, rapid and easy to use when compared with standard identification methods . The 29 species tested were unambiguously separated by carbon source utilization tests . Rhodococcus equi was found to be heterogenous.

J Bacteriol, 1998 Oct, 180(20), 5448 - 53
The 20S proteasome of Streptomyces coelicolor; Nagy I et al.; 20S proteasomes were purified from Streptomyces coelicolor A3(2) and shown to be built from one alpha-type subunit (PrcA) and one beta-type subunit (PrcB) . The enzyme displayed chymotrypsin-like activity on synthetic substrates and was sensitive to peptide aldehyde and peptide vinyl sulfone inhibitors and to the Streptomyces metabolite lactacystin . Characterization of the structural genes revealed an operon-like gene organization (prcBA) similar to Rhodococcus and Mycobacterium spp . and showed that the beta subunit is encoded with a 53-amino-acid propeptide which is removed during proteasome assembly . The upstream DNA region contains the conserved orf7 and an AAA ATPase gene (arc).

Biochem Biophys Res Commun, 1998 Sep 18, 250(2), 364 - 8
Detection of a nitric oxide synthase possibly involved in the regulation of the Rhodococcus sp R312 nitrile hydratase; Sari MA et al.; Crude homogenates from Rhodococcus sp 312 catalyze the conversion of L-arginine into L-citrulline and NO2-, the usual oxidation product of NO under aerobic conditions . They also catalyze the conversion of N omega-hydroxy-L-arginine (NOHA) into L-citrulline and NO2- with similar rates (10-15 and 100-150 nmol of product.min-1.(mg of protein)-1 respectively for the crude homogenate and for a fraction obtained from ammonium sulfate precipitation) . L-citrulline formation is strongly inhibited by classical inhibitors of mammalian nitric oxide synthases (NOSs) such as N omega-methyl-L-arginine (NMA) and thio-L-citrulline (TC) . Finally, the lack of inhibitory effects of EGTA, a classical inhibitor of constitutive mammalian NOSs, and the specific immunodetection of a 100 kD protein from Rhodococcus cytosol by an antibody raised against human inducible NOS, is in favor of the presence of a NOS similar to inducible mammalian NOSs in Rhodococcus sp 312 . This NOS should be responsible for the NO-dependent inactivation of Rhodococcus Nitrile Hydratase (NHase) in the absence of light; it could regulate the activity of the latter enzyme.

Prikl Biokhim Mikrobiol, 1998 Jul-Aug, 34(4), 370 - 6
{Development of microbiological technology of air deodoration in laboratory-industrial conditions using a pilot plant}; Zhukov VG et al.; Laboratory tests were performed to select a complex of bacterial strains capable of effective deodoration of waste air produced by an animal formulated feed works under elevated temperature with the presence of numerous organic pollutants . The complex included species from the general Nocardia, Rhodococcus, and Comamonas . The biocatalyst was tested in a real industrial process with the use of a pilot plant for microbiological deodoration of waste air . The test lasted for over six months and confirmed the efficiency of the development method of deodoration.

J Bacteriol, 1998 Oct, 180(19), 5052 - 7
Limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14 belongs to a novel class of epoxide hydrolases; van der Werf MJ et al.; An epoxide hydrolase from Rhodococcus erythropolis DCL14 catalyzes the hydrolysis of limonene-1,2-epoxide to limonene-1,2-diol . The enzyme is induced when R . erythropolis is grown on monoterpenes, reflecting its role in the limonene degradation pathway of this microorganism . Limonene-1,2-epoxide hydrolase was purified to homogeneity . It is a monomeric cytoplasmic enzyme of 17 kDa, and its N-terminal amino acid sequence was determined . No cofactor was required for activity of this colorless enzyme . Maximal enzyme activity was measured at pH 7 and 50 degrees C . None of the tested inhibitors or metal ions inhibited limonene-1,2-epoxide hydrolase activity . Limonene-1,2-epoxide hydrolase has a narrow substrate range . Of the compounds tested, only limonene-1,2-epoxide, 1-methylcyclohexene oxide, cyclohexene oxide, and indene oxide were substrates . This report shows that limonene-1,2-epoxide hydrolase belongs to a new class of epoxide hydrolases based on (i) its low molecular mass, (ii) the absence of any significant homology between the partial amino acid sequence of limonene-1,2-epoxide hydrolase and amino acid sequences of known epoxide hydrolases, (iii) its pH profile, and (iv) the inability of 2-bromo-4'-nitroacetophenone, diethylpyrocarbonate, 4-fluorochalcone oxide, and 1, 10-phenanthroline to inhibit limonene-1,2-epoxide hydrolase activity.

FEBS Lett, 1998 Aug 14, 433(1-2), 58 - 62
XAS characterization of the active sites of novel intradiol ring-cleaving dioxygenases: hydroxyquinol and chlorocatechol dioxygenases; Briganti F et al.; The intradiol cleaving dioxygenases hydroxyquinol 1,2-dioxygenase (HQI,20) from Nocardiodes simplex 3E, chlorocatechol 1,2-dioxygenase (CIC1,20) from Rhodococcus erythropolis ICP, and their anaerobic substrate adducts (hydroxyquinol-HQ1,20 and 4-chlorocatechol-CIC1,20) have been characterized through X-ray absorption spectroscopy . In both enzymes the iron(III) is pentacoordinated and the distance distribution inside the Fe(III) first coordination shell is close to that already found in the extensively characterized protocatechuate 3,4-dioxygenase . The coordination number and the bond lengths are not significantly affected by the substrate binding . Therefore it is confirmed that the displacement of a protein donor upon substrate binding has to be considered a general step valid for all intradiol dioxygenases.

Respiration, 1998, 65(4), 327 - 30
Rhodococcus equi lung abscess complicating Evan's syndrome treated with corticosteroid; Tsang KW et al.; We report a case of Rhodococcus equi, an unusual pathogen, causing a right upper lobe lung abscess in a patient with Evan's syndrome (auto-immune haemolytic anaemia and thrombocytopenia) who was treated with high-dose corticosteroid therapy . The patient was treated successfully with clarithromycin, vancomycin, ciprofloxacin and imipenen which appear to be effective in combination for this unusual condition in which the treatment regimen has been controversial.

J Basic Microbiol, 1998, 38(3), 207 - 20
Key enzymes for the degradation of benzoate, m- and p-hydroxybenzoate by some members of the order Actinomycetales; Hammann R et al.; A preliminary screening of numerous species of the order Actinomycetales, especially of the genera Mycobacterium, Nocardia, Rhodococcus, Pseudonocardia, and Streptomyces, showed that many of them are able to metabolize benzoate (B) and p-hydroxybenzoate (pHB) as indicated by growth and change of color of the pH-indicator of an agar medium . Subsequent experiments with liquid cultures which allowed the analysis of substrate utilization by thin layer chromatography confirmed these results . The study of the degradative pathway proved that B was metabolized via catechol (C), pHB via protocatechuate (P) and m-hydroxybenzoate (mHB) via gentisate (G) . The aromatic ring of C and P was subjected to an ortho-cleavage; only one strain of Noc . asteroides degraded C via a meta-cleavage, but P via an ortho-cleavage . Cell free extracts of four selected organisms exhibited activity of C-1,2-dioxygenase (C-1,2-O) and/or P-3,4-dioxygenase (P-3,4-O), depending on the growth substrate used for precultivation . In Streptomyces C-1,2-O was only found in cells grown on B, and P-3,4-O only in cells grown on pHB . On the contrary, in Rhodococcus rhodochrous B-cells oxidized C as well as P, while P-cells possessed only P-3,4-O-activity.

Appl Environ Microbiol, 1998 Sep, 64(9), 3270 - 4
Initial transformations in the biodegradation of benzothiazoles by Rhodococcus isolates; De Wever H et al.; Benzothiazole-2-sulfonate (BTSO3) is one of the side products occurring in 2-mercaptobenzothiazole (MBT) production wastewater . We are the first to isolate an axenic culture capable of BTSO3 degradation . The isolate was identified as a Rhodococcus erythropolis strain and also degraded 2-hydroxybenzothiazole (OBT) and benzothiazole (BT), but not MBT, which was found to inhibit the biodegradation of OBT, BT, and BTSO3 . In anaerobic resting cell assays, BTSO3 was transformed into OBT in stoichiometric amounts . Under aerobic conditions, OBT was observed as an intermediate in BT breakdown and an unknown compound transiently accumulated in several assays . This product was identified as a dihydroxybenzothiazole . Benzothiazole degradation pathways seem to converge into OBT, which is then transformed further into the dihydroxy derivative.

Appl Microbiol Biotechnol, 1998 Jul, 50(1), 93 - 7
Cloning and expression of a gene encoding cyanidase from Pseudomonas stutzeri AK61; Watanabe A et al.; The gene coding for cyanidase, which catalyzes the hydrolysis of cyanide to formate and ammonia, was cloned from chromosomal DNA of Pseudomonas stutzeri AK61 into Escherichia coli . The cyanidase gene consisted of an open reading frame of 1004 bp, and it was predicted that cyanidase was composed of 334 amino acids with a calculated molecular mass of 37,518 Da . The amino acid sequence of cyanidase showed a 35.1% and 26.4% homology to aliphatic nitrilase from Rhodococcus rhodochrous K22 and cyanide hydratase from Fusarium lateritium, respectively . A unique cysteine residue of aliphatic nitrilase, which was suggested to play an essential role in the catalytic activity, was conserved in cyanidase . The active form of cyanidase was successfully expressed by a DNA clone containing the cyanidase gene in E . coli . Its productivity was approximately 230 times larger than that of P . stutzeri AK61 . The characteristics of the expressed cyanidase, including optimum pH, optimum temperature . Michaelis constant (K(m)) for cyanide and specific activity, were similar to those of the native enzyme from P . stutzeri AK61.

J Am Vet Med Assoc, 1998 Aug 15, 213(4), 510 - 5
Associations between physical examination, laboratory, and radiographic findings and outcome and subsequent racing performance of foals with Rhodococcus equi infection: 115 cases (1984-1992); Ainsworth DM et al.; OBJECTIVE: To determine whether physical examination, laboratory, or radiographic abnormalities in foals with Rhodococcus equi infection were associated with survival, ability to race at least once after recovery, or, for foals that survived and went on to race, subsequent racing performance . DESIGN: Retrospective study . ANIMALS: 49 Thoroughbreds and 66 Standardbreds admitted to 1 of 6 veterinary teaching hospitals between 1984 and 1992 in which R equi infection was positively diagnosed . PROCEDURE: Results of physical examination, laboratory testing, and thoracic radiography were reviewed . Indices of racing performance were obtained for foals that recovered and eventually raced and compared with values for the US racing population . RESULTS: 83 (72%) foals survived . Foals that did not survive were more likely to have extreme tachycardia (heart rate > 100 beats/min), be in respiratory distress, and have severe radiographic abnormalities on thoracic radiographs at the time of initial examination than were foals that survived . Clinicopathologic abnormalities were not associated with whether foals did or did not survive . Forty-five of the 83 surviving foals (54%) eventually raced at least once, but none of the factors examined was associated with whether foals went on to race . Racing performance of foals that raced as adults was not significantly different from that of the US racing population . CLINICAL IMPLICATIONS: R equi infection in foals is associated with a decreased chance of racing as an adult; however, foals that eventually go on to race perform comparably to the US racing population.

Clin Infect Dis, 1998 Aug, 27(2), 370 - 5
Report of invasive Rhodococcus equi infections in Taiwan, with an emphasis on the emergence of multidrug-resistant strains; Hsueh PR et al.; 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, Waldenstrom'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 (bioMerieux, Marcy l'Etoile, France), VITEK GPI card (bioMerieux 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.

Biochem Biophys Res Commun, 1998 Aug 10, 249(1), 178 - 81
A novel pathway for the metabolism of caffeine by a mixed culture consortium; Madyastha KM et al.; A new oxidative pathway for the degradation of caffeine(1,3,7-Trimethylxanthine, I) by a mixed culture consisting of strains belonging to the genera Klebsiella and Rhodococcus is presented . The mixed culture does not initiate degradation by N-demethylation either complete or partial, but instead carries out oxidation at the C-8 position resulting in the formation of 1,3,7-trimethyluric acid (TMU, II) which further gets degraded to 3,6,8-trimethylallantoin (TMA, III) . Both TMU and TMA are hitherto not shown to be formed in the microbial system . Further degradation of TMA (III) by caffeine grown cells yields dimethylurea (VII) as one of the metabolites . Oxygen uptake studies indicated that caffeine(I) grown cells oxidized TMU(II), TMA (III), glyoxalic acid (VI), dimethylurea(VII), and monomethylurea(V), but not monomethyl and dimethyluric acids . The mixed culture does not accept theophylline(1,3-dimethylxanthine), theobromine(3,7-dimethylxanthine), and paraxanthine(1,7-dimethylxanthine) as the carbon source.

Diagn Cytopathol, 1998 Aug, 19(2), 98 - 101
Multivesiculated macrophages: their implication in fine-needle aspiration cytology of lung mass lesions; Reyes CV et al.; Alveolar macrophages are almost invariably present in percutaneous fine-needle aspiration cytology of the lung . They may predominate, appear foamy and finely vesiculated, or may reflect the cellular composition of the lung mass lesion . In a review of 172 cases of "negative for malignant cells" from the percutaneous lung fine-needle aspiration cytology file in an 8-year period at Hines VA Hospital, the vacuolated macrophages were evaluated qualitatively and quantitatively . Among the 53 cases (34%) showing vacuolated macrophages, only 5-25% of the cells were multivesiculated, the cytoplasmic vacuoles were few, focal, and occasionally global, and the majority of the vacuolated macrophages contained anthracotic or hemosiderin pigments . One case exhibited striking multivesiculation in at least 95% of macrophages and also in bronchial and alveolar cells, fibroblasts, and endothelial and inflammatory cells, a finding consistent with amiodarone toxicity (index case 1) . The diagnosis was confirmed on subsequent transbronchial lung biopsy . In another patient with clinical HIV infection, the multivesiculation was also seen in 95% of the macrophages with associated acute inflammatory exudate, coccobacilli, and a positive culture for Rhodococcus equi (index case 2) . In most cases, the vacuolated macrophages are reactive and inflammatory . Occasionally, as in our index cases they may actually indicate a specific diagnosis.

Microb Comp Genomics, 1998, 3(2), 141 - 8
Sequence analysis of the cupin gene family in Synechocystis PCC6803; Dunwell JM; The recently described cupin superfamily of proteins includes the germin and germinlike proteins, of which the cereal oxalate oxidase is the best characterized . This superfamily also includes seed storage proteins, in addition to several microbial enzymes and proteins with unknown function . All these proteins are characterized by the conservation of two central motifs, usually containing two or three histidine residues presumed to be involved with metal binding in the catalytic active site . The present study on the coding regions of Synechocystis PCC6803 identifies a previously unknown group of 12 related cupins, each containing the characteristic two-motif signature . This group comprises 11 single-domain proteins, ranging in length from 104 to 289 residues, and includes two phosphomannose isomerases and two epimerases involved in cell wall synthesis, a member of the pirin group of nuclear proteins, a possible transcriptional regulator, and a close relative of a cytochrome c551 from Rhodococcus . Additionally, there is a duplicated, two-domain protein that has close similarity to an oxalate decarboxylase from the fungus Collybia velutipes and that is a putative progenitor of the storage proteins of land plants.

Microbiology, 1998 Jul, 144 ( Pt 7), 1981 - 8
Mycobacterial linear plasmids have an invertron-like structure related to other linear replicons in actinomycetes; Picardeau M et al.; The authors previously identified large plasmids in Mycobacterium xenopi, M . branderi and M . celatum which appeared to have a linear topology . This study has confirmed the presence of such linear plasmids in mycobacteria, including M . avium, and demonstrated that the ends of these replicons are covalently bound with protein(s), suggesting an invertron-like structure . The termini of one 25 kb plasmid, designated pCLP, from M . celatum were cloned and the first 500 bp of each terminus were sequenced . The termini of this plasmid show the characteristic features of invertrons with terminal inverted repeats of 45 bp (with imperfect matches) and several palindromic sequences . Moreover, similarity existed in the structure and terminal nucleotide sequence of pCLP and the termini of linear replicons of Streptomyces and Rhodococcus species, indicating a conservation of these linear extrachromosomal elements within the Actinomycetales.

Res Vet Sci, 1998 May-Jun, 64(3), 181 - 5
Pheno- and genotyping of Rhodococcus equi isolated from faeces of healthy horses and cattle; Soedarmanto I et al.; The present study was designed to comparatively investigate 21 Rhodococcus equi isolates from the faeces of clinically healthy horses and cattle . The isolates were identified by cultural and biochemical properties and by PCR analysis . The latter, targeted to the gene coding for the 16S ribosomal RNA, revealed a species specific PCR product . The isolates were further characterised by serotyping with two typing systems, by haemagglutination tests and by plasmid and virulence protein profiling . Among the 21 cultures, four cultures contained plasmids, two of the four cultures expressed 15-17 kDa virulence proteins, no cultures contained 20 kDA virulence proteins . The 21 cultures were further analysed by DNA-fingerprinting . This was performed by macrorestriction analysis of chromosomal DNA by pulsed-field gel electrophoresis (PFGE) . The DNA-restriction patterns were different for most of the isolates indicating a clone heterogeneity among isolates from single farms . Serotyping, determination of virulence marker and PFGE analysis of R equi appeared to be useful for further characterisation of this species, possibly of importance for virulence estimation of single R equi isolates and for epidemiological studies.

Appl Environ Microbiol, 1998 Aug, 64(8), 2931 - 6
Degradation of 1,3-dichloropropene by pseudomonas cichorii 170; Poelarends GJ et al.; The gram-negative bacterium Pseudomonas cichorii 170, isolated from soil that was repeatedly treated with the nematocide 1, 3-dichloropropene, could utilize low concentrations of 1, 3-dichloropropene as a sole carbon and energy source . Strain 170 was also able to grow on 3-chloroallyl alcohol, 3-chloroacrylic acid, and several 1-halo-n-alkanes . This organism produced at least three different dehalogenases: a hydrolytic haloalkane dehalogenase specific for haloalkanes and two 3-chloroacrylic acid dehalogenases, one specific for cis-3-chloroacrylic acid and the other specific for trans-3-chloroacrylic acid . The haloalkane dehalogenase and the trans-3-chloroacrylic acid dehalogenase were expressed constitutively, whereas the cis-3-chloroacrylic acid dehalogenase was inducible . The presence of these enzymes indicates that 1, 3-dichloropropene is hydrolyzed to 3-chloroallyl alcohol, which is oxidized in two steps to 3-chloroacrylic acid . The latter compound is then dehalogenated, probably forming malonic acid semialdehyde . The haloalkane dehalogenase gene, which is involved in the conversion of 1,3-dichloropropene to 3-chloroallyl alcohol, was cloned and sequenced, and this gene turned out to be identical to the previously studied dhaA gene of the gram-positive bacterium Rhodococcus rhodochrous NCIMB13064 . Mutants resistant to the suicide substrate 1,2-dibromoethane lacked haloalkane dehalogenase activity and therefore could not utilize haloalkanes for growth . PCR analysis showed that these mutants had lost at least part of the dhaA gene.

Appl Environ Microbiol, 1998 Aug, 64(8), 2844 - 52
Acyl transfer activity of an amidase from Rhodococcus sp . strain R312: formation of a wide range of hydroxamic acids; Fournand D et al.; The enantioselective amidase from Rhodococcus sp . strain R312 was produced in Escherichia coli and was purified in one chromatographic step . This enzyme was shown to catalyze the acyl transfer reaction to hydroxylamine from a wide range of amides . The optimum working pH values were 7 with neutral amides and 8 with alpha-aminoamides . The reaction occurred according to a Ping Pong Bi Bi mechanism . The kinetic constants demonstrated that the presence of a hydrophobic moiety in the carbon side chain considerably decreased the Km(amide) values (e.g., Km(amide) = 0.1 mM for butyramide, isobutyramide, valeramide, pivalamide, hexanoamide, and benzamide) . Moreover, very high turnover numbers (kcat) were obtained with linear aliphatic amides (e.g., kcat = 333 s-1 with hexanoamide), whereas branched-side-chain-, aromatic cycle- or heterocycle-containing amides were sterically hindered . Carboxylic acids, alpha-amino acids, and methyl esters were not acyl donors or were very bad acyl donors . Only amides and hydroxamic acids, both of which contained amide bonds, were determined to be efficient acyl donors . On the other hand, the highest affinities of the acyl-enzyme complexes for hydroxylamine were obtained with short, polar or unsaturated amides as acyl donors (e.g., KmNH2OH = 20, 25, and 5 mM for acetyl-, alanyl-, and acryloyl-enzyme complexes, respectively) . No acyl acceptors except water and hydroxylamine were found . Finally, the purified amidase was shown to be L-enantioselective towards alpha-hydroxy- and alpha-aminoamides.

Appl Environ Microbiol, 1998 Aug, 64(8), 2800 - 5
A glutathione S-transferase with activity towards cis-1, 2-dichloroepoxyethane is involved in isoprene utilization by Rhodococcus sp . strain AD45; van Hylckama Vlieg JE et al.; Rhodococcus sp . strain AD45 was isolated from an enrichment culture on isoprene (2-methyl-1,3-butadiene) . Isoprene-grown cells of strain AD45 oxidized isoprene to 3,4-epoxy-3-methyl-1-butene, cis-1, 2-dichloroethene to cis-1,2-dichloroepoxyethane, and trans-1, 2-dichloroethene to trans-1,2-dichloroepoxyethane . Isoprene-grown cells also degraded cis-1,2-dichloroepoxyethane and trans-1, 2-dichloroepoxyethane . All organic chlorine was liberated as chloride during degradation of cis-1,2-dichloroepoxyethane . A glutathione (GSH)-dependent activity towards 3, 4-epoxy-3-methyl-1-butene, epoxypropane, cis-1,2-dichloroepoxyethane, and trans-1,2-dichloroepoxyethane was detected in cell extracts of cultures grown on isoprene and 3,4-epoxy-3-methyl-1-butene . The epoxide-degrading activity of strain AD45 was irreversibly lost upon incubation of cells with 1,2-epoxyhexane . A conjugate of GSH and 1, 2-epoxyhexane was detected in cell extracts of cells exposed to 1, 2-epoxyhexane, indicating that GSH is the physiological cofactor of the epoxide-transforming activity . The results indicate that a GSH S-transferase is involved in the metabolism of isoprene and that the enzyme can detoxify reactive epoxides produced by monooxygenation of chlorinated ethenes.

Arch Microbiol, 1998 Aug, 170(2), 85 - 90
Isolation and characterization of a bacterium possessing a novel aldoxime-dehydration activity and nitrile-degrading enzymes; Kato Y et al.; A bacterial strain capable of utilizing E-pyridine-3-aldoxime as a nitrogen source was isolated from soil after a 4-month acclimation period and was identified as Rhodococcus sp . The strain contained a novel aldoxime dehydration activity that catalyzed a stoichiometric dehydration of E-pyridine-3-aldoxime to form 3-cyanopyridine . The enzyme activity was induced by various aldoximes and nitriles . The strain metabolized the aldoxime as follows: E-pyridine-3-aldoxime was dehydrated to form 3-cyanopyridine, which was converted to nicotinamide by a nitrile hydratase, and the nicotinamide was successively hydrolyzed to nicotinic acid by an amidase.

Biochem J, 1998 Aug 1, 333 ( Pt 3), 741 - 7
Purification and characterization of catechol 1,2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 and cloning and sequencing of its catA gene; Strachan PD et al.; A method was developed for the purification of catechol 1, 2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 that had been grown in the presence of benzyl alcohol . The enzyme has very similar apparent Km (1-2 microM) and Vmax (13-19 units/mg of protein) values for the intradiol cleavage of catechol, 3-methylcatechol and 4-methylcatechol and it is optimally active at pH9 . Cross-linking studies indicate that the enzyme is a homodimer . It contains 0.6 atoms of Fe per subunit . The enzyme was crystallized with 15% (w/v) poly(ethylene glycol) 4000/0.33 M CaCl2/25 mM Tris (pH7.5) by using a microseeding technique . Preliminary X-ray characterization showed that the crystals are in space group C2 with unit-cell dimensions a=111.9 A, b=78.1 A, c=134.6 A, beta=100 degrees . An oligonucleotide probe, made by hemi-nested PCR, was used to clone the gene encoding catechol 1,2-dioxygenase (catA) . The deduced 282-residue sequence corresponds to a protein of molecular mass 31539 Da, close to the molecular mass of 31558 Da obtained by electrospray MS of the purified enzyme . catA was subcloned into the expression vector pTB361, allowing the production of catechol 1,2-dioxygenase to approx . 40% of the total cellular protein . The deduced amino acid sequence of the enzyme has 56% and 75% identity with the catechol 1, 2-dioxygenases of Arthrobacter mA3 and Rhodococcus erythropolis AN-13 respectively, but less than 35% identity with intradiol catechol and chlorocatechol dioxygenases of Gram-negative bacteria.

Crit Rev Microbiol, 1998, 24(2), 99 - 147
Biocatalytic sulfur removal from fuels: applicability for producing low sulfur gasoline; McFarland BL et al.; Environmental regulations are driving R&D efforts to produce low sulfur fuels, including diesel fuel and gasoline for motor vehicles . Biocatalytic sulfur removal from fuels has potential applicability for producing low sulfur gasoline . Microbial biocatalysts have been identified that can biotransform sulfur compounds found in fuels, including ones that selectively remove sulfur from dibenzothiophene heterocyclic compounds . Most attention is give to the 4S pathway of Rhodococcus, which can remove sulfur from substituted and unsubstituted dibenzothiophenes, including sulfur compounds that hinder chemical catalysis and that resist removal by mild hydrotreatment . Various bioreactor and bioprocess designs are being tested for use with biocatalysts, including recombinant biocatalysts, for use in removing sulfur from fuels and feedstocks within the petroleum refinery stream . With bioprocess improvements that enhance biocatalyst stability, achieve faster kinetics, improve mass transfer limitations, temperature and solvent tolerance, as well as broaden substrate specificity to attack a greater range of heterocyclic compounds, biocatalysis may be a cost-effective approach to achieve the production of low sulfur gasoline . The challenge will be to accomplish these improvements by the time the regulations for low sulfur gasoline and other vehicle fuels go into effect in order to be competitive with emerging nonbiological desulfurization technologies.

J Appl Microbiol, 1998 May, 84(5), 769 - 76
Involvement of a rhamnolipid-producing strain of Pseudomonas aeruginosa in the degradation of polycyclic aromatic hydrocarbons by a bacterial community; Arino S et al.; A rhamnolipid-producing strain of Pseudomonas aeruginosa GL1 was isolated from a bacterial community growing on a mixture of polycyclic aromatic hydrocarbons (PAH) as sole carbon source . Strain GL1 did not grow on PAH but grew on known degradation metabolites of phenanthrene (O-phthalic acid) and of naphthalene (salicylic acid) . In co-culture with a phenanthrene-degrading strain, Ps . aeruginosa GL1 accelerated the degradation of phenanthrene . Strain GL1 was resistant to toxic amphiphilic compounds such as cationic and anionic detergents . Rhamnolipid production took place in a late stage growth in cultures of strain GL1 on glycerol or n-hexadecane . It coincided with a substantial decrease in cell hydrophobicity and with morphological changes of the outer membrane as observed by transmission electronic microscopy . The rhamnolipids produced inhibited the growth of bacteria such as Rhodococcus erythropolis, Bacillus cereus and Ps . fluorescens . The overall results suggested an outer membrane origin for the rhamnolipids . They also indicate that the utilization of PAH metabolites by strain GL1 is important for the stability of the PAH-degrading community.

J Bacteriol, 1998 Jul, 180(14), 3503 - 8
Characterization of the maleylacetate reductase MacA of Rhodococcus opacus 1CP and evidence for the presence of an isofunctional enzyme; Seibert V et al.; Maleylacetate reductases (EC 1.3.1.32) have been shown to contribute not only to the bacterial catabolism of some usual aromatic compounds like quinol or resorcinol but also to the degradation of aromatic compounds carrying unusual substituents, such as halogen atoms or nitro groups . Genes coding for maleylacetate reductases so far have been analyzed mainly in chloroaromatic compound-utilizing proteobacteria, in which they were found to belong to specialized gene clusters for the turnover of chlorocatechols or 5-chlorohydroxyquinol . We have now cloned the gene macA, which codes for one of apparently (at least) two maleylacetate reductases in the gram-positive, chlorophenol-degrading strain Rhodococcus opacus 1CP . Sequencing of macA showed the gene product to be relatively distantly related to its proteobacterial counterparts (ca . 42 to 44% identical positions) . Nevertheless, like the known enzymes from proteobacteria, the cloned Rhodococcus maleylacetate reductase was able to convert 2-chloromaleylacetate, an intermediate in the degradation of dichloroaromatic compounds, relatively fast and with reductive dehalogenation to maleylacetate . Among the genes ca . 3 kb up- and downstream of macA, none was found to code for an intradiol dioxygenase, a cycloisomerase, or a dienelactone hydrolase . Instead, the only gene which is likely to be cotranscribed with macA encodes a protein of the short-chain dehydrogenase/reductase family . Thus, the R . opacus maleylacetate reductase gene macA clearly is not part of a specialized chlorocatechol gene cluster.

Anal Biochem, 1998 Jul 1, 260(2), 117 - 27
Characterization of proteins utilized in the desulfurization of petroleum products by matrix-assisted laser desorption ionization time-of-flight mass spectrometry; Wolf BP et al.; Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF/MS) with delayed extraction is utilized in linear, reflected-ion and post-source decay (PSD) modes to directly characterize enzymes being developed for use in a petroleum desulfurization process . The DNA sequence for the genes isolated from Rhodococcus sp . strain IGTS8 that produce three of the four enzymes under study had been previously reported with a discrepancy in residue assignments for one of the enzymes, dsz-C . The use of proteolytic digests followed by MALDI/TOF/MS with delayed extraction in the reflected-ion mode provided sequence-specific information with mass accuracies exceeding 40 ppm over a range of masses and signal-to-noise values . Peptide mapping of >80% of the residues was accomplished for all four proteins . The use of PSD established the true sequence for dsz-C, resolving the discrepancy in the literature . A posttranslational loss of N-terminal methionine was observed for each of the four proteins in linear MALDI/MS and was reconfirmed by peptide mapping for three of the proteins .

FEMS Immunol Med Microbiol, 1998 May, 21(1), 11 - 7
Rhodococcus equi infection of monocytes/macrophages from human immunodeficiency virus (HIV)-infected patients and healthy individuals: evaluation of intracellular killing and nitric oxide production; Vullo V et al.; Monocytes/macrophages from human immunodeficiency virus (HIV)-infected patients had a defect in their ability to kill Rhodococeus equi in vitro, as compared with healthy HIV-seronegative individuals . Virulent and avirulent R . equi strains isolated from humans and horses showed no significant intracellular replicative differences within both HIV-positive and -negative monocytes/macrophages . Infection with R . equi induced the production of nitric oxide (NO) by monocytes/macrophages from healthy individuals, but not by cells from HIV-positive patients . The NO formation was significantly inhibited by L-NG-monomethyl arginine and arginase . However . neither competitive inhibition of NO synthesis from L-arginine with L-NMMA nor depletion of arginine with arginase altered the killing activity of human monocytes/macrophages against R . equi, thus suggesting that L-arginine:NO pathway is not required for the intracellular antirhodococcal mechanisms of human monocytes/macrophages.

Appl Microbiol Biotechnol, 1998 May, 49(5), 568 - 72
Overexpression of high-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1 in recombinant Rhodococcus cells; Mizunashi W et al.; High-molecular-mass nitrile hydratase (H-NHase, 530 kDa) is a cobalt-containing enzyme produced by Rhodococcus rhodochrous J1 . For efficient production of H-NHase in R . rhodochrous ATCC12674, several plasmids were constructed . The enzyme was produced in the recombinant Rhodococcus cells only in the presence of an upstream region (approximately 4 kb) of the H-NHase gene under the control of the promoter for the amidase-NHase gene cluster from Rhodococcus sp . N-774 . Although H-NHase was produced as a soluble protein in the cells, the protein did not show NHase activity . However, when the recombinant R . rhodochrous ATCC12674 cells were cultured in the presence of amide compounds, such as crotonamide and methacrylamide, markedly high NHase activity was detected, Gel-filtration chromatography revealed that the NHases produced by the cells grown in the presence and absence of the amide compounds had a molecular mass of more than 500 kDa and 50-80 kDa respectively . These results suggest that the amide compounds are essential for subunit assembly to form an enzymatically active multimer . By the use of the recombinant expression system, NHase activity 1.7 times higher than that of the original strain, R . rhodochrous J1, was achieved.

Appl Environ Microbiol, 1998 Jul, 64(7), 2578 - 84
Biodegradation of variable-chain-length alkanes at low temperatures by a psychrotrophic Rhodococcus sp; Whyte LG et al.; The psychorotrophic Rhodococcus sp . strain Q15 was examined for its ability to degrade individual n-alkanes and diesel fuel at low temperatures, and its alkane catabolic pathway was investigated by biochemical and genetic techniques . At 0 and 5 degrees C, Q15 mineralized the short-chain alkanes dodecane and hexadecane to a greater extent than that observed for the long-chain alkanes octacosane and dotriacontane . Q15 utilized a broad range of aliphatics (C10 to C21 alkanes, branched alkanes, and a substituted cyclohexane) present in diesel fuel at 5 degrees C . Mineralization of hexadecane at 5 degrees C was significantly greater in both hydrocarbon-contaminated and pristine soil microcosms seeded with Q15 cells than in uninoculated control soil microcosms . The detection of hexadecane and dodecane metabolic intermediates (1-hexadecanol and 2-hexadecanol and 1-dodecanol and 2-dodecanone, respectively) by solid-phase microextraction-gas chromatography-mass spectrometry and the utilization of potential metabolic intermediates indicated that Q15 oxidizes alkanes by both the terminal oxidation pathway and the subterminal oxidation pathway . Genetic characterization by PCR and nucleotide sequence analysis indicated that Q15 possesses an aliphatic aldehyde dehydrogenase gene highly homologous to the Rhodococcus erythropolis the A gene . Rhodococcus sp . strain Q15 possessed two large plasmids of approximately 90 and 115 kb (shown to mediate Cd resistance) which were not required for alkane mineralization, although the 90-kb plasmid enhanced mineralization of some alkanes and growth on diesel oil at both 5 and 25 degrees C.

Vet Microbiol, 1998 Mar 15, 61(1-2), 59 - 69
Detection of virulent Rhodococcus equi in tracheal aspirate samples by polymerase chain reaction for rapid diagnosis of R . equi pneumonia in foals; Takai S et al.; Polymerase chain reaction (PCR)-based assays were developed to detect virulent Rhodococcus equi in transtracheal aspirate samples from sick foals showing respiratory signs . An oligonucleotide primer pair from the sequence of the virulence-associated 15- to 17-kDa antigen gene of the virulence plasmid in virulent R . equi was used to amplify a 564 bp region by PCR, and the result was confirmed by Southern blot hybridization . No positive reaction was seen in DNA from 13 different microorganisms typically found in the respiratory tract . In tracheal aspirates seeded with virulent R . equi, a visible band could detect 10 to 10(2) bacteria per PCR assay (10(3) to 10(4)/ml of the aspirate) . Virulent R . equi was demonstrated in 31 of 42 transtracheal aspirates by culture and colony blot analysis, whereas a positive PCR result was observed in only 12 of the 31 culture positive samples . To prevent false-negative results, two methods were developed: a nested PCR and a PCR in combination with enrichment cultures of aspirates in the selective medium to increase the number of bacteria to 10(4)/ml or more . All of the PCR-negative and culture-positive samples were positive by the two methods . These results indicated that PCR-based assays provide a specific and sensitive means to detect virulent R . equi in tracheal aspirates of foals, and they are more rapid than the routine culture procedures for the diagnosis of R . equi pneumonia in foals.

Nat Biotechnol, 1996 Dec, 14(13), 1705 - 9
Molecular mechanisms of biocatalytic desulfurization of fossil fuels; Gray KA et al.; The development of biocatalytic desulfurization of petroleum fractions may allow its use in place of conventional hydrodesulfurization (HDS) . Dibenzothiophene (DBT) is representative of a broad range of sulfur heterocycles found in petroleum that are recalcitrant to desulfurization via HDS . Rhodococcus sp . strain IGTS8 has the ability to convert DBT to 2-hydroxybiphenyl (HBP) with the release of inorganic sulfur . The conversion of DBT to HBP is catalyzed by a multienzyme pathway consisting of two monooxygenases and a desulfinase . The final reaction catalyzed by the desulfinase appears to be the rate limiting step in the pathway . Each of the enzymes has been purified to homogeneity and their kinetic and physical properties studied . Neither monooxygenase has a tightly bound cofactor and each requires an NADH-FMN oxidoreductase for activity . An NADH-FMN oxidoreductase has been purified from Rhodococcus and is a protein of approximately 25,000 molecular weight with no apparent sequence homology to any other protein in the databases . We describe a unique sulfur acquisition system that Rhodococcus uses to obtain sulfur from very stable heterocyclic molecules.

Microbiology, 1998 May, 144 ( Pt 5), 1271 - 9
The terminal structures of linear plasmids from Rhodococcus opacus; Kalkus J et al.; The telomers of several linear plasmids of Rhodococcus opacus (formerly Nocardia opaca) were studied . The plasmids pHG201, pHG204 and pHG205 carry proteins bound to their ends, as shown by gel retardation experiments . A sequence hybridizing with the terminal sequence of pHG207, a recombinant linear plasmid consisting of the left part of pHG204 and the right part of pHG205, which was analysed in a previous study by the authors, could be detected in all linear plasmids of the wild-type R . opacus strains MR11 and MR22 . However, only pHG204 and pHG206 carry terminal inverted repeats (TIRs) like pHG207 . Cloning and sequencing of the terminal fragment of pHG204 revealed a nearly perfect TIR of 1016 bp . In contrast, the termini of pHG201 and pHG205 share little homology . Sequence analysis of the two end fragments of pHG201 revealed a similarity of only 65% within the terminal 34/32 bp and a perfect TIR of only 3 bp . The results support the assumption that long TIRs are not absolutely necessary for replication and maintenance of linear plasmids.

Appl Environ Microbiol, 1998 Jun, 64(6), 2006 - 12
Two nearly identical aromatic compound hydrolase genes in a strong polychlorinated biphenyl degrader, Rhodococcus sp . strain RHA1; Yamada A et al.; The two 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD) hydrolase genes, etbD1 and etbD2, were cloned from a strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp . strain RHA1, and their nucleotide sequences were determined . The etbD2 gene was located in the vicinity of bphA gene homologs and encoded an enzyme whose amino-terminal sequence was very similar to the amino-terminal sequence of the HOHD hydrolase which was purified from RHA1 . Using the etbD2 gene fragment as a probe, we cloned the etbD1 gene encoding the purified HOHD hydrolase by colony hybridization . Both genes encode a product having 274 amino acid residues and containing the nucleophile motif conserved in alpha/beta hydrolase fold enzymes . The deduced amino acid sequences were quite similar to the amino acid sequences of the products of the single-ring aromatic hydrolase genes, such as dmpD, cumD, todF, and xylF, and not very similar to the amino acid sequences of the products of bphD genes from PCB degraders, including RHA1 . The two HOHD hydrolase genes and the RHA1 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HPDA) hydrolase gene, bphD, were expressed in Escherichia coli, and their relative enzymatic activities were examined . The product of bphD was very specific to HPDA, and the products of etbD1 and etbD2 were specific to HOHD . All of the gene products exhibited poor activities against the meta-cleavage product of catechol . These results agreed with the results obtained for BphD and EtbD1 hydrolases purified from RHA1 . The three hydrolase genes exhibited similar induction patterns both in an RNA slot blot hybridization analysis and in a reporter gene assay when a promoter probe vector was used . They were induced by biphenyl, ethylbenzene, benzene, toluene, and ortho-xylene . Strain RCD1, an RHA1 mutant strain lacking both the bphD gene and the etbD2 gene, grew well on ethylbenzene . This result suggested that the etbD1 gene product is involved in the meta-cleavage metabolic pathway of ethylbenzene.

FEMS Microbiol Lett, 1998 May 1, 162(1), 47 - 52
Transfer of plasmid pTO1 from Escherichia coli to various representatives of the order Actinomycetales by intergeneric conjugation; Voeykova T et al.; Plasmid pTO1 containing the oriT fragment from RK2, the Escherichia coli replication function from pBR322, and a DNA fragment of actinophage phi C31 with the attachment site was transferred from E . coli S17-1 to strains of the genera Actinomadura, Arthrobacter, Micromonospora, Nocardia, Rhodococcus, and to 16 strains of the genus Streptomyces . The frequency of conjugant formation was 1 x 10(-3)-1 x 10(-5) depending on the strain . Hybridization experiments demonstrated that plasmid pTO1 integrates into chromosomes of a number of the recipient strains examined.

Curr Microbiol, 1998 May, 36(5), 302 - 8
Pulsed-field gel electrophoresis analysis of the genome of Rhodococcus fascians: genome size and linear and circular replicon composition in virulent and avirulent strains; Pisabarro A et al.; Total DNA of virulent and avirulent strains of Rhodococcus fascians was resolved by pulsed-field gel electrophoresis (PFGE) into a discrete number of fragments by digestion with the endonucleases AseI and DraI . Restriction endonucleases PacI, PmeI, and SwaI yielded no fragments upon digestion of R . fascians genome, and all the other tested endonucleases recognizing 6 bp released too many fragments . The genome size was 5.6 megabases for the type strain R . fascians DSM 20669, and 5.8 megabases for the virulent R . fascians D188 strain . However the genome size of R . fascians CECT 3001 (NRRL B15096) was 8.0 megabases . No linear chromosome in the megabase range was observed under pulse conditions in which Saccharomyces cerevisiae and Schizosaccharomyces pombe chromosomes were perfectly resolved, suggesting that the R . fascians chromosome is circular . A new linear plasmid pIRN640 of 640 kb was found in the avirulent R . fascians CECT 3001 that did not hybridize with a probe internal to the fas region of pFiD188 known to be involved in plant pathogenicity in the virulent strain R . fascians D188 . Virulence was correlated in all strains tested with the presence of the fas region . The AseI and DraI bands corresponding to the extrachromosomal elements were identified providing the basis for a physical map of this organism.

Microbiology, 1998 Apr, 144 ( Pt 4), 955 - 63
Cloning of new Rhodococcus extradiol dioxygenase genes and study of their distribution in different Rhodococcus strains; Kulakov LA et al.; Four extradiol dioxygenase genes which encode enzymes active against catechol and substituted catechols were cloned from two different Rhodococcus strains, and their nucleotide sequences were determined . A catechol 2,3-dioxygenase gene (edoC) was shown to be identical to the previously described ipbC gene from the isopropylbenzene operon of Rhodococcus erythropolis . Amino acid sequences deduced from the three other genes (edoA, edoB and edoD) were shown to have various degrees of homology to different extradiol dioxygenases . The EdoA and EdoB dioxygenases were classified as belonging to the third family of type I oxygenases and represented two new subfamilies, whereas the EdoD dioxygenase was a type II enzyme . Analysis of six Rhodococcus strains revealed a wide distribution of the above dioxygenase genes . Rhodococcus sp . 11 was shown to harbour all four of the analysed dioxygenase genes . Nucleotide sequences homologous to the edoB gene were present in all of the strains, including R . erythropolis NCIMB 13065, which did not utilize any of the aromatic compounds analysed . The latter finding points to the existence of a silent pathway(s) for degradation of aromatic compounds in this Rhodococcus strain.

Infect Immun, 1998 May, 66(5), 1848 - 54
Cytokine induction in murine macrophages infected with virulent and avirulent Rhodococcus equi; Giguere S et al.; To look for a possible correlation between the virulence of Rhodococcus equi and its cytokine-inducing capacity, we evaluated intracellular survival and measured cytokine induction by mouse macrophages infected with a virulent strain containing an 85-kb plasmid and expressing VapA (103+), its avirulent plasmid-cured derivative (103-), and heat-killed 103+ (HK) . After incubation with similar numbers of bacteria, macrophages infected with 103- contained significantly more organisms than those infected with 103+ or HK . The number of bacteria in the macrophages infected with 103- and HK decreased progressively, whereas the 103+ numbers remained constant over 48 h . Interleukin 1beta (IL-1beta), IL-6, IL-10, IL-12 p40, and tumor necrosis factor alpha (TNF-alpha) mRNA induction peaked at 4 h and returned to baseline between 12 and 48 h postinfection . IL-1beta, IL-6, IL-10, and TNF-alpha concentrations assessed by enzyme-linked immunosorbent assay generally agreed well with mRNA expression; IL-12 could, however, not be detected . For all the cytokines detected, mean concentrations in the supernatants were consistently higher in the 103(-)-infected monolayers than in those infected with 103+, although, with the exception of IL-1beta, the differences were not statistically significant . R . equi HK was a poor inducer of cytokine production . In conclusion, virulent and avirulent R . equi strains induced similar levels of cytokine synthesis . The slightly greater induction of most cytokines observed following infection with 103- is likely secondary to greater uptake by macrophages rather than to a direct role of VapA or another plasmid-encoded product in downregulating cytokine induction.

Scand J Infect Dis, 1997, 29(6), 535 - 41
Rhodococcus equi pneumonia in patients infected with the human immunodeficiency virus . Report of 2 cases and review of the literature; Capdevila JA et al.; Rhodococcus equi is a cause of lung infection in immunosuppressed hosts . Since the start of the HIV epidemic, 76 cases of R . equi lung infection (MEDLINE 1985-96) affecting this population have been described . We report 2 additional cases and review the clinical data, radiological findings, treatment and outcome of these 78 patients . The mean age of these patients was 33 y; 69 were male . 71 met the criteria for AIDS (CDC 1993) . Fever and cough were the presenting complaints in the majority of patients (84.3%) . A single cavitary lung lesion in the upper lobes was the most common radiological finding (57.7%), although multiple cavitations, alveolar infiltrates and pleural effusion were also found . Treatment usually was based on synergistic antibiotic combinations for a long period of time determined on an individual basis . Surgery was performed only in 11 patients . Death attributable directly to R . equi infection is low (15.4%), however only half of the patients (53.8%) were completely cured . We conclude that R . equi infection should be strongly considered in any HIV patient who presents with cavitary lesions in the lung, especially if mycobacteria are not identified . Treatment must be based on synergistic antibiotic combinations, and surgery relegated to cases of chronic single cavitary lesions not responding to antibiotics.

J Mol Biol, 1998 Mar 20, 277(1), 13 - 25
Characterization of ARC, a divergent member of the AAA ATPase family from Rhodococcus erythropolis; Wolf S et al.; A gene encoding a AAA ATPase was discovered in the 5' region of the second operon of 20 S proteasome subunits in the nocardioform actinomycete Rhodococcus erythropolis NI86/21 . The gene was cloned and expressed in Escherichia coli . The protein, ARC (AAA ATPase forming Ring-shaped Complexes), is a divergent member of the AAA family . The deduced product of the arc gene is 591 residues long (66 kDa) . The purified protein possesses a low, N-ethylmaleimide-sensitive ATPase activity and forms rings of six subunits, arranged symmetrically around a central opening or cavity . Two-dimensional crystals grown on lipid monolayers yielded images of the ATPase molecules in "end-on" orientation at 1.9 nm resolution .

Appl Environ Microbiol, 1998 Apr, 64(4), 1276 - 82
Characterization of the rrnB operon of the plant pathogen Rhodococcus fascians and targeted integrations of exogenous genes at rrn loci; Pisabarro A et al.; A 6.0-kb SalI DNA fragment containing an entire rRNA operon (rrnB) was cloned from a cosmid gene bank of the phytopathogenic strain Rhodococcus fascians D188 . The nucleotide sequence of the 6-kb fragment was determined and had the organization 16S rRNA-spacer-23S rRNA-spacer-5S rRNA without tRNA-encoding genes in the spacer regions . The 5' and 3' ends of the mature 16S, 23S, and 5S rRNAs were determined by alignment with the rrn operons of Bacillus subtilis and other gram-positive bacteria . Four copies of the rrn operons were identified by hybridization with an rrnB probe in R . fascians type strain ATCC 12974 and in the virulent strain R . fascians D188 . However, another isolate, CECT 3001 (= NRRL B15096), also classified as R . fascians, produced five rrn-hybridizing bands . An integrative vector containing a 2.5-kb DNA fragment internal to rrnB was constructed for targeted integration of exogenous genes at the rrn loci . Transformants carrying the exogenous chloramphenicol resistance gene (cmr) integrated in different rrn operons were obtained . These transformants had normal growth rates in complex medium and minimal medium and were fully stable for the integrated marker.

J Am Vet Med Assoc, 1998 Apr 1, 212(7), 976 - 81
Physical and serologic examinations of foals at 30 and 45 days of age for early diagnosis of Rhodococcus equi infection on endemically infected farms; Higuchi T et al.; OBJECTIVE: To evaluate results of physical and serologic examinations of foals at 30 and 45 days of age on 3 types of farms with various prevalences of clinical disease (endemic, sporadic, none) caused by Rhodococcus equi and to determine whether evaluations were helpful in early diagnosis and control of the disease . DESIGN: Prospective cohort study . ANIMALS: 144 foals at 30 and 45 days of age . PROCEDURE: During a 2-year period, 36 foals on farms at which R equi infection was endemic, 71 foals on farms at which the disease was sporadically detected, and 37 foals on farms without the disease were examined by means of auscultation of lungs, serum biochemical and hematologic analyses, and determination of antibody titers against R equi, using ELISA . Transtracheal aspirates were obtained from 14 of 32 foals that had clinical signs of disease and 7 of 41 seropositive foals that did not have clinical signs of disease . RESULTS: Prevalences of respiratory tract disease and seropositive conversion rates for 45-day-old foals on endemically and sporadically infected farms were significantly higher than on farms without the disease . Rhodococcus equi was isolated from tracheal aspirates of seropositive foals, even when clinical signs were not evident . CLINICAL IMPLICATIONS: Physical and serologic examinations of foals at 30 and 45 days of age were useful for early diagnosis of R equi infection, especially for foals on farms at which the disease was endemic.

J Vet Med Sci, 1998 Feb, 60(2), 277 - 9
Improved electroporation of Rhodococcus equi; Sekizaki T et al.; The condition of an electroporation method was re-evaluated for the introduction of foreign plasmid DNA into Rhodococcus equi . The method is based on an electroporation of the bacteria made competent by culturing in a broth containing glycine and by heat shock at 50 degrees C . Transformation of R . equi could be achieved with a chloramphenicol-resistant shuttle vector originating from Rhodococcus fascians at an efficiency of about 10(4) transformants/microgram DNA . The bacteria were also shown to become competent when they were incubated with a chemical transformation buffer prior to washing with an electroporation buffer.

Clin Infect Dis, 1998 Mar, 26(3), 749 - 52
Vertebral osteomyelitis due to Rhodococcus equi in a liver transplant recipient; Fischer L et al.; 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.

Eur J Clin Microbiol Infect Dis, 1998 Jan, 17(1), 55 - 7
Thyroid abscess due to Rhodococcus equi in a patient infected with the human immunodeficiency virus; Martin-Davila P et al.; A case of thyroid abscess due to Rhodococcus equi in an HIV-positive patient with previous pulmonary abscess is reported . Rhodococcus equi is a gram-positive rod that can cause infections in both immunocompetent and immunocompromised patients, though it occurs more frequently in patients with dysfunctional cellular immune systems . Several cases of Rhodococcus equi infection in persons infected with HIV have been reported . In these patients Rhodococcus equi usually invades the lungs, producing pneumonia . These infections often relapse, accompanied by intermittent bacteremia, despite conventional treatment . Extrapulmonary abscesses can occur.

J Bacteriol, 1998 Mar, 180(5), 1082 - 94
Evolutionary relationship between chlorocatechol catabolic enzymes from Rhodococcus opacus 1CP and their counterparts in proteobacteria: sequence divergence and functional convergence; Eulberg D et al.; Biochemical investigations of the muconate and chloromuconate cycloisomerases from the chlorophenol-utilizing strain Rhodococcus opacus (erythropolis) 1CP had previously indicated that the chlorocatechol catabolic pathway of this strain may have developed independently from the corresponding pathways of proteobacteria . To test this hypothesis, we cloned the chlorocatechol catabolic gene cluster of strain 1CP by using PCR with primers derived from sequences of N termini and peptides of purified chlorocatechol 1,2-dioxygenase and chloromuconate cycloisomerase . Sequencing of the clones revealed that they comprise different parts of the same gene cluster in which five open reading frames have been identified . The clcB gene for chloromuconate cycloisomerase is transcribed divergently from a gene which codes for a LysR-type regulatory protein, the presumed ClcR . Downstream of clcR but separated from it by 222 bp, we detected the clcA and clcD genes, which could unambiguously be assigned to chlorocatechol 1,2-dioxygenase and dienelactone hydrolase . A gene coding for a maleylacetate reductase could not be detected . Instead, the product encoded by the fifth open reading frame turned out to be homologous to transposition-related proteins of IS1031 and Tn4811 . Sequence comparisons of ClcA and ClcB to other 1,2-dioxygenases and cycloisomerases, respectively, clearly showed that the chlorocatechol catabolic enzymes of R . opacus 1CP represent different branches in the dendrograms than their proteobacterial counterparts . Thus, while the sequences diverged, the functional adaptation to efficient chlorocatechol metabolization occurred independently in proteobacteria and gram-positive bacteria, that is, by functionally convergent evolution.

J Bacteriol, 1998 Mar, 180(5), 1072 - 81
Characterization of a protocatechuate catabolic gene cluster from Rhodococcus opacus 1CP: evidence for a merged enzyme with 4-carboxymuconolactone-decarboxylating and 3-oxoadipate enol-lactone-hydrolyzing activity; Eulberg D et al.; The catechol and protocatechuate branches of the 3-oxoadipate pathway, which are important for the bacterial degradation of aromatic compounds, converge at the common intermediate 3-oxoadipate enol-lactone . A 3-oxoadipate enol-lactone-hydrolyzing enzyme, purified from benzoate-grown cells of Rhodococcus opacus (erythropolis) 1CP, was found to have a larger molecular mass under denaturing conditions than the corresponding enzymes previously purified from gamma-proteobacteria . Sequencing of the N terminus and of tryptic peptides allowed cloning of the gene coding for the 3-oxoadipate enol-lactone hydrolase by using PCR with degenerate primers . Sequencing showed that the gene belongs to a protocatechuate catabolic gene cluster . Most interestingly, the hydrolase gene, usually termed pcaD, was fused to a second gene, usually termed pcaC, which encodes the enzyme catalyzing the preceding reaction, i.e., 4-carboxymuconolactone decarboxylase . The two enzymatic activities could not be separated chromatographically . At least six genes of protocatechuate catabolism appear to be transcribed in the same direction and in the following order: pcaH and pcaG, coding for the subunits of protocatechuate 3,4-dioxygenase, as shown by N-terminal sequencing of the subunits of the purified protein; a gene termed pcaB due to the homology of its gene product to 3-carboxy-cis,cis-muconate cycloisomerases; pcaL, the fused gene coding for PcaD and PcaC activities; pcaR, presumably coding for a regulator of the IclR-family; and a gene designated pcaF because its product resembles 3-oxoadipyl coenzyme A (3-oxoadipyl-CoA) thiolases . The presumed pcaI, coding for a subunit of succinyl-CoA:3-oxoadipate CoA-transferase, was found to be transcribed divergently from pcaH.

Kekkaku, 1998 Jan, 73(1), 37 - 42
{The 72nd Annual Meeting Education Lecture . Cord factor}; Yano I; Mycobacterial cell wall contains various lipids (glycolipids and phospholipids) to contribute to its hydrophobic property or acid-fastness and these surface molecules contact with host cells in the early step of infection . Among them, cord factor (trehalose 6,6'-dimycolate, TDM or CF) is the most ubiquitous component, which may be a key molecule for pathogenesis and immunity . Initially, cord factor was isolated from a highly virulent strain of M . tuberculosis which grows in the form of serpentine cords, and showed a marked toxicity for mice when it was administrated intravenously . These observations led to the early hypothesis that cell wall components are related to virulence . However, later studies revealed that cord factors were also found in other non-cord-forming mycobacterial species and other mycolic acid-containing bacteria . Structural studies demonstrated that there were various mycoloyl glycolipids differing in carbohydrate moiety such as glucose mycolate, mannose mycolate, arabinose mycolate and fructose mycolate besides trehalose mycolate in acid-fast bacteria . Therefore, the interest has been focused to the structure-activity relationships of mycoloyl glycolipid and to the mechanism of virulence for host animals . So far, it has been demonstrated that cord factor showed lethal toxicity, granuloma forming activity, adjuvant activity, tumor regressing activity and non-specific infection prevention activity in experimental animals . We have extended investigations further on the structure analysis and immunomodifying activities of cord factor and related mycoloyl glycolipids from various species of mycobacteria, nocardia, rhodococci and gordona, and demonstrated that the most activities were shown in trehalose or glucose esters, but not in mannose, arabinose or fructose esters . Furthermore, it was shown that the longer chain-mycoloyl glycolipids showed the higher toxicity and immunomodifying activities . In mice, in vivo, cytokine inducing activities such as IL-1, IFN-gamma, TNF-alpha, GM-CSF and chemotactic factor were observed and in vitro, TNF-alpha, GM-CSF, chemotactic factor, complement, NO, PGE2 inductions and protein kinase C activation were demonstrated . Furthermore, recently, we have demonstrated that cord factor induced a marked thymic atrophy due to the cortical lymphocyte apoptosis before granuloma formation in mice . It was also established that cord factor showed antigenicity in mice and rabbits and human tuberculous patient sera contained specific antibody (IgG) reactive against cord factor . From above results, cord factor seems to be one of the most potent immunomodulators in the mycobacterial cell wall components pathologically and beneficially.

Transplantation, 1998 Feb 15, 65(3), 449 - 53
Rhodococcus equi infection in transplant recipients: case report and review of the literature; Munoz P et al.; BACKGROUND: Rhodococcus equi is an opportunistic pathogen that usually causes infection in immunocompromised hosts, mainly human immunodeficiency virus-positive patients, yet solid organ transplant recipients may be affected as well . Infections in this group of patients have not been sufficiently analyzed . METHODS: We report an R equi pneumonia in a heart transplant recipient and review another 11 cases . RESULTS: Infection appeared a mean of 49 months (range 1-180) after transplantation . Lung was primarily involved in 10 cases (83.3%) . The remaining two cases presented with a paravertebral abscess and a purulent pericarditis . Invasive techniques were necessary to reach the diagnosis in nine cases . One patient healed with surgical resection of the lesion; the remaining 11 received antimicrobial agents . Six of them required additional surgical treatment . Three patients died . CONCLUSIONS: Clinicians should consider R equi when evaluating a solid organ recipient with an asymptomatic lung nodule . Microbiology laboratories should be alerted in these cases because it could be mistaken for a contaminant diphtheroid and will not respond to the standard empirical therapy.

Prikl Biokhim Mikrobiol, 1997 Jul-Aug, 33(4), 383 - 7
{Isolation of nitrile hydratase from Rhodococcus rhodochrous M8 cells and determination of the N-terminal amino acid sequence of its subunits}; Sinolitskii MK et al.; Nitrile hydratase was isolated and purified to homogeneity from cells of Rhodococcus rhodochrous M8 . This enzyme catalyzes the hydrolysis of acrylic acid nitrile to acrylamide . Nitrile hydratase content in the cell was shown to be 17% of total soluble protein . The molecular weight of the native enzyme was 510 kDa . The enzyme consisted of two subunits with molecular weights of 23.5 kDa and 28.0 kDa . The N-terminal amino acid sequences of these subunits were estimated.

J Vet Med Sci, 1997 Dec, 59(12), 1097 - 101
Isolation of virulent Rhodococcus equi from transtracheal aspirates of foals serodiagnosed by enzyme-linked immunosorbent assay; Higuchi T et al.; Although isolation of Rhodococcus equi from tracheobronchial aspirates is thought to be a definitive diagnosis of R . equi pneumonia in foals, virulence of isolates from the aspirates of infected foals remains obscure . In the present study, transtracheal aspirates were collected from thirty-one 1- to 6-month-old foals, which showed clinical signs of respiratory tract infection, and R . equi isolates were analyzed for the presence of virulence plasmids and virulence-associated antigens . Moreover, this method was compared with a serodiagnosis by an enzyme-linked immunosorbent assay (ELISA) to evaluate the sensitivity of the ELISA . Of the 31 foals, 21 revealed positive cultures for R . equi . Of the 21 foals, 20 (95%) had an ELISA OD value of 0.3 (positive limit of this test) or higher at the initial medical examination . All of the isolates from the aspirates were virulent R . equi, which contained virulence plasmids and expressed virulence-associated antigens . In the remaining 10 foals showing a negative culture for R . equi, 3 foals had positive ELISA titers . Six foals died during the treatment, and necropsy revealed that 5 of the 6 foals had R . equi infection characterized by large abscesses in the lungs, and 3 of the 5 foals also had intestinal lesions . All clinical isolates from the lesions of the foals were virulent R . equi . These results support the assumption that isolates from the transtracheal aspirates of infected foals are virulent R . equi and the sensitivity of ELISA might demonstrate a serodiagnostic value for early diagnosis of R . equi infection in foals.

Res Immunol, 1997 Jul-Aug, 148(6), 387 - 97
T-cell response during Rhodococcus equi infection in a murine experimental model; Matsiota-Bernard P et al.; Rhodococcus equi is a facultative intracellular bacterium that can cause pneumonia in both young horses and immunocompromised humans . In this study, we have tried to determine the T-cell populations that recognize this pathogen during murine infection, as well as the bacterial antigens recognized by these cells . When BALB/c mice were hyperimmunized with a virulent R . equi strain, we did not observe preferential expansion of a particular T-cell subset in their spleens . However, when the splenic T lymphocytes of the hyperimmunized BALB/c mice were cultured in the presence of killed bacteria, we found that alpha/beta CD4+ T cells proliferated and exhibited increased levels of the interleukin-2 receptor (IL2R) . In order to ensure antigen specificity, two different controls were included in these experiments: (i) T-cell proliferation and expression of the IL2R in the presence of the major membrane constituent of Bacillus megaterium were studied comparatively with the presence of the R . equi bacterial antigen, and (ii) T-cell proliferation and expression of the IL2R from naive, non-infected mice in the presence of bacterial antigens were compared to those observed in hyperimmunized mice . In our study, the T cells from hyperimmunized mice did not significantly proliferate nor were they activated in the presence of non-related bacterial antigens, and T cells from naive mice were not found to significantly recognize R . equi antigens . When we studied the localization of R . equi antigens that could stimulate the in vitro proliferation and activation of T cells, we found that they were constituents of the bacterial cell wall and the cytoplasm, but they were not excreted in the culture medium . For these experiments, T-cell recognition of the bacterial antigens in hyperimmunized mice was compared to that of naive mice . With T-cell immunoblotting, we found that T-cell proliferation and activation were obtained with proteins having molecular masses of approximately 65, 43, 30, 22-27 and 15-17 kDa . It is noteworthy that among the recognized bacterial antigens, some have been described as being associated with virulence.

Zentralbl Bakteriol, 1997 Nov, 286(4), 457 - 67
Identification and epidemiological relationship of Rhodococcus equi isolated from cases of lymphadenitis in cattle; Soedarmanto I et al.; The present study was designed to comparatively investigate 10 Rhodococcus equi isolates from cases of lymphadenitis in cattle . The isolates could be identified by cultural and biochemical properties . By serotyping the R . equi isolates 9 and 1, cultures could be classified as Nakazawa's serotypes 15 and 8, respectively . The isolates did not agglutinate rabbit erythrocytes, were uniformly susceptible to most of the antibiotics tested, did not contain plasmids nor expressed virulence-associated proteins and yielded identical patterns in protein fingerprinting . To further analyze the epidemiological relationships, the isolates were additionally subjected to DNA fingerprinting . This was performed by pulsed-field gel electrophoresis (PFGE) after digestion of the chromosomal DNA with the endonuclease AsnI . PFGE analysis of the chromosomal DNA revealed 4 DNA restriction groups with DNA pattern I with 7 isolates as predominant group and DNA pattern II to IV with one isolate, respectively . The present results indicate that a single R . equi clone belonging to Nakazawa's serotype 15 and according to PFGE to DNA restriction pattern I of the present investigation seems to be responsible for most of the cases of lymphadenitis of cattle described in this study.

Plasmid, 1997, 38(3), 180 - 7
Development of a Rhodococcus equi-Escherichia coli plasmid shuttle vector; Zheng H et al.; 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.

Biotechnol Appl Biochem, 1997 Dec, 26 ( Pt 3), 159 - 62
Nutritional factors that affect the production of cholesterol oxidase by Rhodococcus equi no . 23; Lee MT et al.; Some nutritional factors affecting the production of cholesterol oxidase (COX) by Rhodococcus equi no . 23 were investigated . Cholesterol and yeast extract respectively were the best carbon source and nitrogen source for the COX production . The optimum concentration of cholesterol and yeast extract was found to be about 0.1% and 0.4-0.5% (w/v) respectively . In addition, NH4Cl, NaCl and Tween 80 also exhibited enhancing effects on COX production, being maximal at 0.1% (w/v), 0.2% (w/v) and 1.0% (v/v) respectively . Moreover, optimal enzyme production occurred in medium that had an initial pH of 7.0.

FEBS Lett, 1997 Nov 24, 418(1-2), 189 - 94
Dissecting the assembly pathway of the 20S proteasome; Zuhl F et al.; Proteasomes reach their mature active state via a complex cascade of folding, assembly and processing events . The Rhodococcus proteasome offers a means to dissect the assembly pathway and to characterize intermediates; its four subunits (alpha1, alpha2, beta1, beta2) assemble efficiently in vitro with any combination of alpha and beta . Assembly studies with wild-type and N-terminally truncated beta-subunits in conjunction with refolding studies allowed to define the role of the propeptide which is two-fold: It supports the initial folding of the beta-subunits and it promotes the maturation of the holoproteasomes.

J Biol Chem, 1997 Nov 21, 272(47), 29454 - 9
Structure of the photoreactive iron center of the nitrile hydratase from Rhodococcus sp . N-771 . Evidence of a novel post-translational modification in the cysteine ligand; Tsujimura M et al.; Nitrile hydratase (NHase) from Rhodococcus sp . N-771 is a photoreactive enzyme that is inactivated by nitrosylation of the non-heme iron center and activated by photodissociation of nitric oxide (NO) . To obtain structural information on the iron center, we isolated peptide complexes containing the iron center by proteolysis . When the tryptic digest of the alpha subunit isolated from the inactive form was analyzed by reversed-phase high performance liquid chromatography, the absorbance characteristic of the nitrosylated iron center was observed in the peptide fragment, Asn105-Val-Ile-Val-Cys-Ser-Leu-Cys-Ser-Cys-Thr-Ala-Trp-Pro-Ile-Leu - Gly-Leu-Pro-Pro-Thr-Trp-Tyr-Lys128 . The peptide contained 0.79 mol of iron/mol of molecule as well as endogenous NO . Subsequently, by digesting the peptide with thermolysin, carboxypeptidase Y, and leucine aminopeptidase M, we found that the minimum peptide segment required for the nitrosylated iron center is the 11 amino acid residues from alphaIle107 to alphaTrp117 . Furthermore, by using mass spectrometry, protein sequence, and amino acid composition analyses, we have shown that the 112th Cys residue of the alpha subunit is post-translationally oxidized to a cysteine-sulfinic acid (Cys-SO2H) in the NHase . These results indicate that the NHase from Rhodococcus sp . N-771 has a novel non-heme iron enzyme containing a cysteine-sulfinic acid in the iron center . Possible ligand residues of the iron center are discussed.

J Bacteriol, 1997 Dec, 179(24), 7776 - 83
Cloning and expression of a gene from Streptomyces scabies encoding a putative pathogenicity factor; Bukhalid RA et al.; We cloned a 9.4-kb DNA fragment from Streptomyces scabies ATCC 41973 that allows the nonpathogen Streptomyces lividans 66 TK24 to necrotize and colonize potato tuber slices and produce scab-like symptoms on potato minitubers . Deletion analysis demonstrated that activity was conferred by a 1.6-kb DNA region . Sequence analysis of a 2.4-kb DNA fragment spanning the DNA region necessary for activity revealed three open reading frames (ORFs) . The deduced amino acid sequence of ORF1, designated ORFtnp, showed high levels of identity with the first 233 amino acids of the putative transposases of the IS1164 elements from Rhodococcus rhodochrous (71%) and Mycobacterium bovis (68%), members of the Staphylococcus aureus IS256 family of transposases . No significant homologies to ORF2 and ORF3 were found in the nucleic acid and protein databases . ORFtnp is located 5' of ORF3 . ORF2 is incomplete and is located 3' of ORF3 . Subcloning of the individual ORFs demonstrated that ORF3, designated nec1, is sufficient for necrotizing activity in S . lividans 66 TK24 . S . lividans 66 TK24 expressing nec1 does not produce thaxtomin A but produces an unidentified extracellular water-soluble compound that causes necrosis on potato tuber discs . The G+C content of nec1 suggests that it has moved horizontally from another genus . Southern analysis of ORFtnp and nec1 demonstrate that these genes are physically linked in Streptomyces strains, including S . scabies and Streptomyces acidiscabies strains, that are pathogenic on potato and that produce the phytotoxin thaxtomin A . These data suggest that nec1 may have been mobilized into S . scabies through a transposition event mediated by ORFtnp.

Eur J Biochem, 1997 Nov 1, 249(3), 739 - 47
Two-component flavin-dependent pyrrole-2-carboxylate monooxygenase from Rhodococcus sp; Becker D et al.; Pyrrole-2-carboxylate can serve as the sole source of carbon, nitrogen, and energy for a strain tentatively identified to belong to the genus Rhodococcus . An NADH-dependent oxygenase activity was detected in cell extracts that initiated the degradation of the substrate . During purification of the enzyme, this activity was separated into two protein components which were both purified to apparent homogeneity . A small monomeric 18.7-kDa protein designated as reductase, catalyzed in vitro the NADH and FAD-dependent reduction of cytochrome c and had an NADH-oxidase activity . The second component, a 54-kDa protein with a trimeric native structure had no enzymatic activity by itself, but exhibited a pyrrole-2-carboxylate-dependent oxygen consumption when it was complemented with the reductase component, FAD, and NADH . This indicated that the large protein referred to as oxygenase was responsible for the oxygen-dependent hydroxylation of the substrate . The rate of an uncoupled NADH oxidation without hydroxylation of the substrate was found to be strongly dependent on the molar ratio of both components . The uncoupling was nearly completely suppressed by a 5-7-fold molar excess of the oxygenase component . The small protein was N-terminally blocked . It was thus proteolytically digested and four of the resulting peptides were sequenced comprising 47 amino acids . The sequences of these fragments were similar to the sequences reported for the small component of different two-component flavin monooxygenases . Furthermore, the N-terminus of the oxygenase component showed high sequence similarity to the second, usually large subunit of these enzymes and to two single-component flavin monooxygenases . Thus, the enzyme from Rhodococcus sp . designated as pyrrole-2-carboxylate monooxygenase belongs to the recently discovered new class of two-component flavin aromatic monooxygenases . Some of the basic properties of both components were determined and their interaction during catalysis was investigated.

Acta Cytol, 1997 Nov-Dec, 41(6), 1833 - 8
Pulmonary malakoplakia diagnosed by fine needle aspiration . A case report; Lambert C et al.; BACKGROUND: Pulmonary malakoplakia is an uncommon disorder, with 24 previously reported cases, only 4 of which were diagnosed by bronchial washings, bronchial brushings or aspiration cytology . We report a case that was diagnosed initially by computed tomography (CT)-guided fine needle aspiration (FNA) cytology . CASE: A 56-year-old male with follicular small cleaved cell lymphoma had a 10-cm left lower lobe mass compressing the main bronchus to that lobe . A transthoracic, CT-guided FNA specimen consisted predominantly of foamy macrophages, many of which contained typical Michaelis-Gutmann bodies . Microbiologic cultures identified Rhodococcus equi . A subsequent transbronchial biopsy and left pneumonectomy specimen confirmed the cytologic diagnosis . CONCLUSION: Pulmonary malakoplakia associated with R equi pneumonia is a rare lesion that is essentially limited to immunocompromised hosts . Awareness of the FNA cytomorphology of this lesion permits resolution of the typical clinical differential diagnosis of pulmonary masses in the immunocompromised host and can facilitate treatment.

J Bacteriol, 1997 Nov, 179(22), 7156 - 60
Designing recombinant Pseudomonas strains to enhance biodesulfurization; Gallardo ME et al.; The dsz biodesulfurization cluster from Rhodococcus erythropolis IGTS8 has been engineered under the control of heterologous broad-host-range regulatory signals to alleviate the mechanism of sulfur repression, and it was stably inserted into the chromosomes of different Pseudomonas strains . The recombinant bacteria were able to desulfurize dibenzothiophene more efficiently than the native host . Furthermore, these new biocatalysts combine relevant industrial and environmental traits, such as production of biosurfactants, with the enhanced biodesulfurization phenotype.

Mol Microbiol, 1997 Sep, 25(5), 989 - 98
Identification and characterization of an aliphatic amidase in Helicobacter pylori; Skouloubris S et al.; We report, for the first time, the presence in Helicobacter pylori of an aliphatic amidase that, like urease, contributes to ammonia production . Aliphatic amidases are cytoplasmic acylamide amidohydrolases (EC 3.5.1.4) hydrolysing short-chain aliphatic amides to produce ammonia and the corresponding organic acid . The finding of an aliphatic amidase in H . pylori was unexpected as this enzyme has only previously been described in bacteria of environmental (soil or water) origin . The H . pylori amidase gene amiE (1017 bp) was sequenced, and the deduced amino acid sequence of AmiE (37746Da) is very similar (75% identity) to the other two sequenced aliphatic amidases, one from Pseudomonas aeruginosa and one from Rhodococcus sp . R312 . Amidase activity was measured as the release of ammonia by sonicated crude extracts from H . pylori strains and from recombinant Escherichia coli strains overproducing the H . pylori amidase . The substrate specificity was analysed with crude extracts from H . pylori cells grown in vitro; the best substrates were propionamide, acrylamide and acetamide . Polymerase chain reaction (PCR) amplification of an internal amiE sequence was obtained with each of 45 different H . pylori clinical isolates, suggesting that amidase is common to all H . pylori strains . A H . pylori mutant (N6-836) carrying an interrupted amiE gene was constructed by allelic exchange . No amidase activity could be detected in N6-836 . In a N6-urease negative mutant, amidase activity was two- to threefold higher than in the parental strain N6 . Crude extracts of strain N6 slowly hydrolysed formamide . This activity was affected in neither the amidase negative strain (N6-836) nor a double mutant strain deficient in both amidase and urease activities, suggesting the presence of an independent discrete formamidase in H . pylori . The existence of an aliphatic amidase, a correlation between the urease and amidase activities and the possible presence of a formamidase indicates that H . pylori has a large range of possibilities for intracellular ammonia production.

Biochemistry (Mosc), 1997 Aug, 62(8), 872 - 82
Site-specific endonucleases RspLKI and RspLKII from Rhodococcus species LK2 are isoschizomers of SphI and BamHI; Zabaznaya EV et al.; Two site-specific endonucleases, RspLKI and RspLKII, have been isolated and purified to functional homogeneity from the soil bacterium Rhodococcus species LK2 . RspLKI recognizes the 5'-GCATG decreases C-3' DNA sequence and RspLKII recognizes the 5'-G decreases GATCC-3' sequence (arrows indicate DNA cleavage sites) . The isolated enzymes are class II site specific endonucleases and are isoschizomers of endonucleases SphI and BamHI, respectively.

APMIS, 1997 Sep, 105(9), 705 - 7
Purulent meningitis due to Rhodococcus equi . A case of posttraumatic infection; Tunger A et al.; Opportunistic infections due to Rhodococcus equi have been increasingly reported in the immunocompromised population, especially in patients with AIDS . In this report, we present an unusual case of purulent meningitis that developed in an immunocompetent six-year-old child through direct inoculation of R . equi.

Proc Natl Acad Sci U S A, 1997 Oct 28, 94(22), 11986 - 91
Identification of active sites in amidase: evolutionary relationship between amide bond- and peptide bond-cleaving enzymes; Kobayashi M et al.; Mainly based on various inhibitor studies previously performed, amidases came to be regarded as sulfhydryl enzymes . Not completely satisfied with this generally accepted interpretation, we performed a series of site-directed mutagenesis studies on one particular amidase of Rhodococcus rhodochrous J1 that was involved in its nitrile metabolism . For these experiments, the recombinant amidase was produced as the inclusion body in Escherichia coli to greatly facilitate its recovery and subsequent purification . With regard to the presumptive active site residue Cys203, a Cys203 --> Ala mutant enzyme still retained 11.5% of the original specific activity . In sharp contrast, substitutions in certain other positions in the neighborhood of Cys203 had a far more dramatic effect on the amidase . Glutamic acid substitution of Asp191 reduced the specific activity of the mutant enzyme to 1.33% of the wild-type activity . Furthermore, Asp191 --> Asn substitution as well as Ser195 --> Ala substitution completely abolished the specific activity . It would thus appear that, among various conserved residues residing within the so-called signature sequence common to all amidases, the real active site residues are Asp191 and Ser195 rather than Cys203 . Inasmuch as an amide bond (CO-NH2) in the amide substrate is not too far structurally removed from a peptide bond (CO-NH-), the signature sequences of various amidases were compared with the active site sequences of various types of proteases . It was found that aspartic acid and serine residues corresponding to Asp191 and Ser195 of the Rhodococcus amidase are present within the active site sequences of aspartic proteinases, thus suggesting the evolutionary relationship between the two.

J Bacteriol, 1997 Oct, 179(19), 6145 - 53
A 3-(3-hydroxyphenyl)propionic acid catabolic pathway in Rhodococcus globerulus PWD1: cloning and characterization of the hpp operon; Barnes MR et al.; Rhodococcus globerulus PWD1, a soil isolate from a polluted site in The Netherlands, is able to degrade a broad range of aromatic compounds . A novel gene cluster which appears to encode a pathway for the degradation of phenolic acids such as 3-(3-hydroxyphenyl)propionate (3HPP) has been cloned from the chromosome of this organism . Sequence analysis of a 7-kb region identified five open reading frames (ORFs) . Analysis of mRNA showed that the genes were expressed during growth on 3HPP and 3-hydroxyphenylacetate (3HPA) but not during growth on m-cresol or succinate . The first ORF, hppA, which appears to be separately transcribed, had considerable amino acid identity with a number of hydroxylases . Transcriptional analysis indicates that the next four ORFs, hppCBKR, which are tightly clustered, constitute a single operon . These genes appear to encode a hydroxymuconic semialdehyde hydrolase (HppC), an extradiol dioxygenase (HppB), a membrane transport protein (HppK), and a member of the IclR family of regulatory proteins (HppR) . The activities of HppB and HppC have been confirmed by enzyme assay of Escherichia coli hosts . The substrate specificity of HppB expressed from the cloned gene matches that of the meta-cleavage dioxygenase expressed from wild-type Rhodococcus grown on both 3HPP and 3HPA and is considerably more active against acid than against neutral catechols . The deduced amino acid sequences of the gene products have a recognizable homology with a broad range of enzymes and proteins involved in biodegradation and appear most similar to the mhp operon from E . coli K-12, which also encodes the degradation of 3HPP.

Can J Microbiol, 1997 Sep, 43(9), 841 - 6
Biodegradation of groundwater pollutants by a combined culture of Mycobacterium vaccae and a Rhodococcus sp; Fairlee JR et al.; The catabolism of selected groundwater pollutants by a combined culture of Mycobacterium vaccae and a Rhodococcus sp . (strain R-22) was investigated . The M . vaccae-R-22 combined culture was five times more effective in mineralizing benzene than either organism alone . Mycobacterium vaccae oxidized benzene to phenol, and R-22 catabolized the phenol to cellular components and CO2 . Benzene did not support growth of M . vaccae, R-22, or the combined culture . Optimization of ratios of the two species indicated that the maximum mineralization of benzene occurred at an initial ratio of 75% M . vaccae to 25% R-22 . Cell fractionation of the combined culture after mineralization of {U-14C}benzene indicated that 10% of the benzene carbon was incorporated into cell material, and of this 45% was present in protein and 20% in nucleic acids . This suggested that minimally one species could utilize the products of benzene as a nutrient source . The M . vaccae-R-22 combined culture catabolized ethylbenzene and chlorobenzene without the accumulation of phenolic intermediates, which are inhibitory to M . vaccae's ability to degrade the parent compounds . This study demonstrates that defined mixed cultures may be useful in studying the effects of environmental pollutant degradation on microbial ecosystems and mineralization of these pollutants by the ecosystem.

J Biol Chem, 1997 Oct 17, 272(42), 26103 - 9
Active site-directed inhibitors of Rhodococcus 20 S proteasome . Kinetics and mechanism; Mc Cormack T et al.; We have studied the mechanism of inhibition of the recombinant Rhodococcus proteasome by four different chemical classes of active site-directed small molecule inhibitors . Clasto-lactacystin beta-lactone is a time-dependent inhibitor of the Rhodococcus proteasome's ability to hydrolyze Suc-Leu-Leu-Val-Tyr-AMC, a substrate for this proteasome's single type of active site, and proceeds with a kinact/{I} of 1,700 M-1 s-1 . Using peptide mapping of tryptic digests, LC/MS, and amino acid sequence analysis, we have established that the Ogamma of the hydroxyl group on the N-terminal threonine of the beta-subunit is the sole modification made by the beta-lactone . Active site titrations of the Rhodococcus proteasome with reversible peptide aldehydes show the expected stoichiometry of one inhibitor molecule per beta-subunit . Prior modification with beta-lactone completely abrogates the binding of peptidyl boronic acid inhibitors, suggesting that these inhibitors also inactivate the enzyme by reacting with the Ogamma moiety on Thr1 . High performance liquid chromatography analysis of peptidyl vinyl sulfone-modified intact Rhodococcus proteasome beta-subunit and its tryptic peptides suggests that the peptidyl vinyl sulfone modifies a residue in the N-terminal 20 amino acids . This modification is also blocked by prior treatment with beta-lactone.

Microbiology, 1997 Sep, 143 ( Pt 9), 2975 - 81
A Rhodococcus species that thrives on medium saturated with liquid benzene; Paje ML et al.; A bacterium isolated from a contaminated site in Sydney, Australia, utilized benzene in the liquid phase as a sole carbon source at levels toxic to other micro-organisms . The organism was a short Gram-positive rod which grew at 6% NaCl, 0-37 degrees C and pH 2-10 . Biochemical tests, fatty acid analysis, and 16S rDNA sequencing identified the organism as a member of the genus Rhodococcus . Vapour-phase addition of benzene to the medium in batch and continuous systems resulted in initial concentrations averaging 200 p.p.m . Under these conditions, 95% of the benzene was degraded . In separate experiments, medium spiked with liquid benzene resulted in concentrations of up to 2789 p.p.m . and supported good growth of the organism . To confirm utilization of benzene at levels known to be toxic to other micro-organisms, continuous cultures were used; benzene added at 2% (v/v) per day resulted in growth and 89% degradation, which was maintained for more than 30 d . Rhodococcus sp . strain 33 appears to be the only organism known that can grow at these levels of benzene.

Microbiology, 1997 Sep, 143 ( Pt 9), 2961 - 73
Elucidation of the metabolic pathway for dibenzothiophene desulphurization by Rhodococcus sp . strain IGTS8 (ATCC 53968); Oldfield C et al.; Rhodococcus sp . strain IGTS8 (ATCC 53968) is able to utilize dibenzothiophene (DBT) as a sole source of sulphur . The carbon skeleton of DBT is not metabolized and is conserved as 2-hydroxybiphenyl (HBP), which accumulates in the medium . This phenotype is due to the expression of the plasmid-encoded DBT-desulphurization (dsz) operon, which encodes three proteins, DszA, B and C . In this paper it is shown, using {35S}DBT radiolabelling studies, that sulphur is released in the form of inorganic sulphite . The pathway of DBT desulphurization is described in detail . In summary, DszC catalyses the stepwise S-oxidation of DBT, first to dibenzothiophene 5-oxide (DBTO) and then to dibenzothiophene 5,5-dioxide (DBTO2); DszA catalyses the conversion of DBTO2 to 2-(2'-hydroxyphenyl)benzene sulphinate (HBPSi-) and DszB catalyses the desulphination of HBPSi- to give HBP and sulphite . Studies with cell-free extracts show that DszA and DszC, but not DszB, require NADH for activity . 18O2-labelling studies show that each incorporated oxygen atom is derived directly from molecular oxygen . These results are consistent with the role of DszC as a mono-oxygenase, of DszA as an apparently unique enzyme which catalyses the reductive hydroxylation of DBTO2 leading to cleavage of the thiophene ring, and of DszB as an aromatic sulphinic acid hydrolase.

Biochem Biophys Res Commun, 1997 Sep 8, 238(1), 197 - 201
Biosynthesis of a cyclic tautomer of (3-methylmaleyl)acetone from 4-hydroxy-3,5-dimethylbenzoate by Pseudomonas sp . HH35 but not by Rhodococcus rhodochrous N75; Cain RB et al.; Here we report that the bacterial catabolism of 4-hydroxy-3,5-dimethylbenzoic acid 1 takes a different course in Rhodococcus rhodochrous N75 and Pseudomonas sp . strain HH35 . The former organism accumulates a degradation metabolite of the acid which we isolated and identified as 2,6-dimethylhydroquinone 2 . The latter bacterial strain converts the acid and the hydroquinone into a dead-end metabolite . This novel compound was characterised unequivocally by mass spectrometry and 1H and 18C NMR and UV spectroscopy as 4-acetonyl-4-hydroxy-2-methylbut-2-en-1,4-olide 4, a cyclic tautomer of (3-methylmaleyl)acetone, which exists as the enol carboxylate form 8 in aqueous solution.

FEMS Microbiol Lett, 1997 Sep 1, 154(1), 95 - 102
Identification and characterization of IS-like elements in Mycobacterium gordonae; Picardeau M et al.; A new insertion sequence, IS1512, from Mycobacterium gordonae was cloned and sequenced . This element is present in up to 10 copies which provides a high diversity for restriction fragment length polymorphism analysis . We have also identified truncated IS1512-like elements, including a truncated IS1512 and another truncated insertion sequence which displays homology with IS1512 and was designated IS1511 . Sequences homologous to the previously described transposon Tn554 are inserted into these truncated insertion sequences . Insertion loci of IS1511/IS1512 are shown to be highly related to those described for IS256-like elements in other species . Alignment of the putative transposases from Rhodococcus and Mycobacterium suggests these insertion sequences may form a distinct closely homologous subclass within the IS256 family . Analysis comparing phylogenetic divergence of these elements with that of 16S rRNA and superoxide dismutase genes suggests horizontal transfer of a IS1511/IS1512 precursor into M . gordonae, and the occurrence of horizontal transfer between Mycobacteriaceae and Rhodococcaceae.

Appl Environ Microbiol, 1997 Sep, 63(9), 3385 - 9
Purification and characterization of limonoate dehydrogenase from Rhodococcus fascians; Humanes L et al.; Limonoate dehydrogenase from Rhodococcus fascians has been purified to electrophoretic homogeneity by a procedure that consists of ion-exchange, hydrophobic, and affinity chromatography . The native enzyme has a molecular mass of around 128,000 Da and appears to be composed of four similar subunits (30,000 Da each) . The isoelectric point is 4.9 as determined by isoelectric focusing . The homogeneous enzyme was used to determine the NH2-terminal amino acid sequence . The enzyme was purified from cells grown in either fructose or limonoate as a carbon source . Limonoate dehydrogenase activity was higher in limonoate-grown cultures . Additionally, the enzyme preparations differed in their affinity for limonoids but not for NAD+ . In all cases limonoate dehydrogenase exhibited a higher catalytic rate and stronger affinity for limonoate A-ring lactone than for disodium limonoate, the limonoid traditionally used for in vitro activity assays . Our data confirm previous reports proposing that limonoate A-ring lactone is the physiological substrate for limonoate dehydrogenase . The increase in limonoate dehydrogenase activity observed in limonoate-grown cultures appears to be caused by a rise in protein levels, since chloramphenicol prevented such an effect.

J Biol Chem, 1997 Aug 8, 272(32), 19846 - 50
Molecular cloning, expression, and sequence analysis of the endoglycoceramidase II gene from Rhodococcus species strain M-777; Izu H et al.; 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.

Biotechnol Appl Biochem, 1997 Aug, 26 ( Pt 1), 19 - 25
Some biochemical properties and the classification of a range of bacterial haloalkane dehalogenases; Damborsky J et al.; Multivariate analyses and experimental data have been used to evaluate the relationships between eight bacterial hydrolytic haloalkane dehalogenases . The results indicate that seven of the dehalogenases investigated can confidently be placed into two Classes {sensu Slater, Bull and Hardman (1995) Biodegradation 6, 181-189} according to their substrate profiles . The remaining enzyme, isolated from Rhodococcus erythropolis CP9, appears to represent a third Class of haloalkane dehalogenases.

South Med J, 1997 Aug, 90(8), 851 - 4
Spontaneous resolution of rhodococcal pulmonary infection in a liver transplant recipient; Schilz RJ et al.; Pulmonary infection by Rhodococcus equi is characterized by indolent infection in an immunocompromised host with a propensity to form cavitary lesions . Mortality can be greater than 50%; treatment involves prolonged therapy with multiple antibiotics and, occasionally, surgical resection . Recurrence is common . We report a case of a liver transplant patient with a pulmonary nodule caused by R equi; the nodule followed a benign clinical course and resolved spontaneously . This case illustrates that the spectrum of disease caused by R equi is not fully appreciated and that significant pitfalls complicate the diagnosis and management of infection by this unusual and probably underrecognized pathogen.

Appl Environ Microbiol, 1997 Aug, 63(8), 3282 - 5
Three of the seven bphC genes of Rhodococcus erythropolis TA421, isolated from a termite ecosystem, are located on an indigenous plasmid associated with biphenyl degradation; Kosono S et al.; Rhodococcus erythropolis TA421, a polychlorinated biphenyl and biphenyl degrader isolated from a termite ecosystem, has seven bphC genes expressing 2,3-dihydroxybiphenyl dioxygenase activity . R . erythropolis TA421 harbored a large and probably linear plasmid on which three (bphC2, bphC3, and bphC4) of the seven bphC genes were located . A non-biphenyl-degrading mutant, designated strain TA422, was obtained spontaneously from R . erythropolis TA421 . TA422 lacked the plasmid, suggesting that the three bphC genes were involved in the degradation of biphenyl . Southern blot analyses showed that R . erythropolis TA421 and Rhodococcus globerulus P6 have a similar set of bphC genes and that the genes for biphenyl catabolism are located on plasmids of different sizes . These results indicated that the genes encoding the biphenyl catabolic pathway in Rhodococcus strains are borne on plasmids.

Zentralbl Veterinarmed B, 1997 Jul, 44(5), 287 - 94
Studies on the rod-coccus life cycle of Rhodococcus equi; Fuhrmann C et al.; In the present study all 19 Rhodococcus equi cultures isolated from horses and 19 of 22 R . equi cultures isolated from human patients displayed a rod-coccus life cycle after cultivation under defined growth conditions . A bacillary growth could be observed after cultivation of the bacteria in fluid media for 4 h at 37 degrees C, a coccoid morphology after cultivation of the bacteria for 24 h either on sheep blood agar plates or in fluid media . The different morphological features did not significantly influence the typability of the bacteria or the expression of surface proteins including 15-17 kDa virulence proteins . Studies on further surface characteristics revealed a relation between haemagglutinating properties, the surface hydrophobicity and adherence properties of the bacteria to HeLa cells . These properties seemed to be influenced by the cultivation conditions but not by the different morphological forms of the bacteria . A haemagglutination reaction, a hydrophobic surface and an enhanced adherence to HeLa cells could be observed with coccoid bacteria after cultivation in fluid media for 24 h at 37 degrees C but not with coccoid bacteria harvested from sheep blood agar plates or with bacillary bacteria after 4 h growth in fluid media . This difference might possibly be caused by the degree of encapsulation of the bacteria after various cultivation conditions and a subsequent masking effect of the hydrophilic polysaccharide capsule of R . equi.

Lett Appl Microbiol, 1997 Jul, 25(1), 75 - 9
New method for RNA isolation from actinomycetes: application to the transcriptional analysis of the alcohol oxidoreductase gene thcE in Rhodococcus and Mycobacterium; Nagy I et al.; 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.

Int J Syst Bacteriol, 1997 Jul, 47(3), 904 - 7
Reclassification of Nocardioides simplex ATCC 13260, ATCC 19565, and ATCC 19566 as Rhodococcus erythropolis; Yoon JH et al.; Our phylogenetic analysis based on 16S ribosomal DNA (rDNA) sequences and chemotaxonomic analyses showed that Nocardioides simplex ATCC 13260, ATCC 19565, and ATCC 19566 are more closely related to the genus Rhodococcus, especially Rhodococcus erythropolis, than to the genus Nocardioides . N . simplex ATCC 13260 and N . simplex ATCC 19565 and ATCC 19566 exhibited levels of 16S rDNA similarity of 99.4 and 100%, respectively, to R . erythropolis DSM 43066T . Strains ATCC 13260, ATCC 19565, and ATCC 19566 had mesodiaminopimelic acid in their peptidoglycan and MK-8(H2) as their predominant menaquinone . These three strains produced cellular fatty acid patterns similar to those of R . erythropolis strains rather than those of Nocardioides species . Therefore, N . simplex ATCC 13260, ATCC 19565, and ATCC 19566 should be reclassified as strains of R . erythropolis Gray and Thornton 1928.

J Bacteriol, 1997 Jul, 179(14), 4635 - 8
Transposition of the IS21-related element IS1415 in Rhodococcus erythropolis; Nagy I et al.; Three copies of the IS21-related transposable element IS1415 were identified in Rhodococcus erythropolis NI86/21 . Adjacent to one of the IS1415 copies, a 47-bp sequence nearly identical to the conserved 5' end of integrons was found . Accurate transposition of IS1415 carrying a chloramphenicol resistance gene (Tn5561) was demonstrated following delivery from a suicide vector to R . erythropolis SQ1.

Appl Environ Microbiol, 1997 Jul, 63(7), 2915 - 9
Conservation of plasmid-encoded dibenzothiophene desulfurization genes in several rhodococci; Denis-Larose C et al.; The cloned sulfur oxidation (desulfurization) genes (sox) for dibenzothiophene (DBT) from the prototype Rhodococcus sp . strain IGTS8 were used in Southern hybridization and PCR experiments to establish the DNA relatedness in six new rhodococcal isolates which are capable of utilizing DBT as a sole sulfur source for growth . The ability of these strains to desulfurize appears to be an exclusive property of a 4-kb gene locus on a large plasmid of ca . 150 kb in IGTS8 and ca . 100 kb in the other strains . Besides a difference in plasmid profile, IGTS8 is distinguishable from the other strains in at least the copy number of the insertion sequence IS1166, which is associated with the sox genes.

J Clin Microbiol, 1997 Jul, 35(7), 1904 - 8
Emergence of rifampin-resistant Rhodococcus equi in an infected foal; Takai S et al.; To investigate the emergence of rifampin resistance in Rhodococcus equi strains isolated from foals and their environment in Japan, we compared the in vitro antimicrobial susceptibilities to rifampin of 640 isolates from 64 infected foals and 98 soil isolates from their horse-breeding farms . As a control, 39 human isolates from patients with and without AIDS were also tested for susceptibility to rifampin . All of the isolates showed rifampin sensitivity, except isolates from one infected foal and two patients with AIDS that showed rifampin resistance . To investigate the emergence of rifampin-resistant R . equi in the infected foal, which had received rifampin monotherapy for a month before euthanasia, 99 isolates of R . equi from the lesions and 20 isolates from the intestinal contents of the one foal with rifampin-resistant organisms were analyzed for rifampin susceptibilities, pathogenicities, and ribotypes . Of the 99 isolates from the lesions, all of which were virulent R . equi strains containing a virulence plasmid with a size of 85 or 90 kb, 90 (91%) isolates were rifampin resistant (MIC, > or = 12.5 microg/ml) . On the other hand, of the 20 isolates from the intestinal contents, 11 (55%) isolates showed rifampin resistance (MIC, > or = 25 microg/ml), and 5 of them were avirulent R . equi strains . Among these 101 rifampin-resistant R . equi isolates with and without virulence plasmids characterized by ribotyping, 58 were type I, 20 were type II, 11 were type III, and 12 were type IV . These results demonstrated that at least eight different rifampin-resistant R . equi strains emerged concurrently and respectively from the different lesions and intestinal contents of the infected foal.

Vet Microbiol, 1997 Jun 16, 56(3-4), 313 - 34
Clinical manifestations, diagnosis, treatment, and prevention of Rhodococcus equi infections in foals; Giguere S et al.; Since the 1986 Rhodococcus equi workshop, there have been major breakthroughs in understanding the epidemiology of, the virulence of, and the immune response to, this intriguing pathogen . However, with the exception of the use of hyperimmune plasma for the prevention of the disease (Martens et al., 1989; Madigan et al., 1991) the clinical aspects of R . equi infections have essentially remained unchanged . This article reviews the various clinical manifestations and summarizes recent advances in diagnosis, treatment and prevention of R . equi infections in foals.

Vet Microbiol, 1997 Jun 16, 56(3-4), 301 - 12
Pathogenicity and virulence of Rhodococcus equi in foals following intratracheal challenge; Wada R et al.; Twelve foals, between 27 and 83 days old, were infected with 2 strains of Rhodococcus equi by intratracheal administration . Ten of the 12 foals were inoculated with 10(4)-10(10) colony forming units (cfu) of ATCC 33701 strain . The other 2 foals were inoculated with 10(9) cfu of a plasmid-cured derivative of the ATCC 33701 strain (ATCC 33701P-) . All of the 10 foals challenged with the ATCC 33701 strain showed clinical signs of pulmonary disease within 5-13 days, such as gross lesions associated with acute bronchopneumonia and microscopic lesions associated with granulomatous pneumonia . The two foals challenged with the ATCC 33701P- strain showed neither clinical signs of disease nor gross lesions . Apparently, when lacking plasmid, the virulent Rhodococcus equi lost its pathogenicity.

Vet Microbiol, 1997 Jun 16, 56(3-4), 287 - 99
Macroamphiphilic cell envelope components of Rhodococcus equi and closely related bacteria; Sutcliffe IC; Recent progress towards an understanding of the architecture of the mycobacterial cell envelope (P.J . Brennan and H . Nikaido, Annual Review of Biochemistry 64 (1995) 29-63) provides a model with features more generally applicable to cell envelope organisation in other mycolic acid-containing bacteria . Using this archetype, a model for the organisation of the rhodococcal cell envelope is presented here, with particular reference to cell envelope composition in Rhodococcus equi . The likelihood that mycolic acids bound to the cell wall arabinogalactan contribute to the formation of a distinct outer lipid layer is emphasised . Furthermore, the model incorporates recent work which has characterised rhodococcal macroamphiphiles (lipoglycans and lipoproteins), including the VapA virulence-associated lipoproteins of R . equi.

Vet Microbiol, 1997 Jun 16, 56(3-4), 277 - 85
In vitro production of tumor necrosis factor-alpha, interleukin-6 and interleukin-8 from normal human peripheral blood mononuclear cells stimulated by Rhodococcus equi; Pece S et al.; The capability of heat-killed Rhodococcus equi organisms to induce in vitro release of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-8 from normal human mononuclear cells as well as the secretion kinetics of these inflammatory cytokines over a 48 h period were evaluated . Results show that normal human mononuclear cells are efficiently triggered to secrete TNF-alpha, IL-6 and IL-8 following R . equi stimulation according to a different kinetics . In particular, release of IL-B was already maximally expressed after 2 h of stimulation, while TNF-alpha amounts progressively increased in a time-dependent fashion . Finally, IL-6 secretion reached peak levels as soon as 18 h of incubation . Taken together, these data point out that monocyte-derived cytokines may play an important role in the immunological control of R . equi infection in immunocompetent people.

Vet Microbiol, 1997 Jun 16, 56(3-4), 257 - 68
Pathogenesis and virulence of Rhodococcus equi; Hondalus MK; Inhalation of the soil-borne organism, Rhodococcus equi, can lead to a chronic and severe pyogranulomatous pneumonia in young horses and immunocompromised people . In addition, ulcerative colitis is a common sequela to infection in foals, and dissemination from the lung to other body sites is not uncommon in either the horse or man . Although the facultative intracellular bacterium is susceptible to neutrophil-mediated killing, it is able to resist innate macrophage defenses and establish residence within the intracellular environment of that phagocyte . Definitive virulence factors of R . equi have not yet been determined, but potential candidates include capsular polysaccharide, the exoenzyme cholesterol oxidase, cell wall mycolic acids, and the products encoded by a virulence-associated plasmid . The ability to replicate within the macrophage is associated with virulence, and correlates in animals with the possession of a large plasmid and expression of the plasmid-encoded, surface-expressed lipoprotein, VapA . All strains of R . equi isolated from horses with clinical disease possess a large plasmid and express VapA antigens . In addition, bacterial clearance and granuloma development in mice is linked to plasmid possession and VapA expression . Plasmid containing strains replicate within the tissues of the mouse . whereas plasmid-cured strains are rapidly cleared . At present, the function of the VapA protein is unknown . In contrast to what is observed in the foal, only a small percentage of R . equi strains isolated from humans with rhodococcal disease express VapA antigens, although a high proportion of others express a related protein which is associated with reduced virulence and is also plasmid-encoded . In a limited number of plasmid-negative human isolates, virulence has been linked to beta-lactam resistance, and preliminary evidence suggests that the phenotype may be phage encoded . It is likely that the immune status of the patient can influence whether a particular strain of R . equi is able to produce clinical disease, and certainly experimental infection in mice has confirmed that an intact cellular immune response is necessary for clearance of the organism.

Vet Microbiol, 1997 Jun 16, 56(3-4), 247 - 55
Antigenic analysis of Rhodococcus equi preparations using different horse sera; Fontanals AM et al.; An R . equi vaccine, prepared under conditions which induce the expression of many antigens, and which has given encouraging results in field trials, was analyzed by SDS-PAGE and immunoblots and compared with other R . equi preparations: a preparation made in with the same technique from a nonvirulent isolate (virulence associated protein negative, VapA-negative); a whole cell preparation of a VapA-positive R . equi, prepared as a standard bacterin; and a semipurified VapA preparation (APTX) . The antigens in these preparations were analyzed using hyperimmune sera (from adult horses vaccinated with the R . equi vaccine), passively and actively immunized foals' sera, asymptomatic but serologically positive foals' sera sera from R . equi pneumonic foals, an equine APTX antiserum, and a VapA monoclonal antibody (Mab) . The vaccine under study had many proteins in high concentrations . Hyperimmune sera reacted strongly with vaccine antigens in the high molecular weight regions . In the low molecular weight range, it reacted in the 14 and less kDa zone . Sera from passively immunized foals reacted similarly but not so strongly . Actively immunized foals gave very weak reactions . With the APTX extract, the Mab reacted with bands at 15-17, 44 and 66 kDa; it reacted weakly with the whole cell and not with the VapA-negative preparations . The APTX antiserum and the Mab reacted strongly with the vaccine at the 14 and less kDa zone, and also with bands at 21, 44 and 66 kDa and very tenuously at 18 kDa, but not in the expected 15-17 kDa zone, suggesting that the native form of VapA is altered but without loss of antigenicity in the vaccine preparation . Our results suggest that other higher molecular weight antigens, in addition to VapA, may be important in inducing antibodies that protect young foals from R . equi pneumonia . These antigens are in high concentrations and in an immunogenic form in the vaccine.

Vet Microbiol, 1997 Jun 16, 56(3-4), 213 - 25
Assessment of the immunogenic potential of Rhodococcus equi virulence associated protein (VapA) in mice; Prescott JF et al.; The development of immunity to Rhodococcus equi, particularly to a virulence-associated protein (VapA) based antigen preparation, was examined in CD1 and BALB/c mice after intraperitoneal vaccination . Immunization with VapA based antigen without adjuvant markedly enhanced organ clearance in CD1 mice but not in BALB/c mice . Delayed type hypersensitivity response and antibody titres in VapA based antigen immunized BALB/c mice were less than in CD1 mice . By contrast also to CD1 mice, sera from immunized BALB/c mice did not react as strongly with VapA in western blots . Use of adjuvants (aluminium hydroxide, iscoms) interfered markedly with the immunogenic properties of the VapA based antigen, in the case of aluminium hydroxide by apparently driving a Th2 type of response . Unexpectedly, iscom adjuvants also impaired immunity and, despite the highest DTH response, produced a low IgG2a response, suggesting that iscomization of the antigen produced a low interferon gamma and high interleukin 2 response . Passive immunization of BALB/c mice with serum from mice immunized with live virulent strain 103+ resulted in only temporary and slight enhancement of organ clearance, supporting the central importance of cellular immunity to R . equi . Immunization with live virulence plasmid- and VapA-positive R . equi strain 103 resulted in marked liver clearance, in marked DTH response and high antibody titres . By contrast, immunization with live virulence plasmid- and VapA-negative strain 103 resulted in slight but variable enhancement of clearance, but insignificant DTH and antibody . The virulence plasmid, and by implication VapA, was thus shown to be critical in determining a highly effective protection to live organisms.

Vet Microbiol, 1997 Jun 16, 56(3-4), 205 - 12
Prevention of Rhodococcus equi pneumonia of foals using two different inactivated vaccines; Varga J et al.; Two different, inactivated, aluminium salt adsorbed vaccines, one containing a R . equi strain (serotype 1, 10(9) CFU/ml and equine herpesvirus 2 (EHV-2) (1.5 x 10(7) PFU/ml) and another containing R . equi only were used on three studfarms to determine whether the disease can be prevented by vaccination of both pregnant mares and their foals . Pregnant mares received two 3 ml doses of vaccine intramuscularly 6 and 2 weeks before parturition and their foals were vaccinated on two or three occasions at 3, 5 or 7 weeks of age . The efficacy of the vaccines was evaluated on the basis of the clinical signs, serological response (indirect haemagglutination and virus neutralisation tests) and culture of R . equi from sick or dead foals . On studs A and B where the bivalent vaccine was used, 24 and 14 foals were born respectively to the vaccinated mares but no clinical case or death occurred due to R . equi pneumonia, while out of the 10 nonvaccinated control foals (stud B) two succumbed to R . equi pneumonia and 4 other foals had to be treated with antibiotics because of fever, coughing and dyspnea . In stud C, where the vaccine containing R . equi strain alone was used, all 15 vaccinated foals remained healthy but one of the 11 control foals died of suppurative R . equi pneumonia and one foal had to be treated due to R . equi pneumonia . R . equi strains (serotype 1) were isolated from the lungs of all dead foals . The serological response was very weak to both R . equi and the EHV-2 strain . Antibody titres in the colostrum of the vaccinated mares against R . equi (in studs A and B, geometric mean 3.79 +/- 1.63 and 4.14 +/- 1.46, respectively) were practically not higher than titres in the controls (in stud B geometric mean 2.12 +/- 1.96) . More antibody was present in the colostrum samples against EHV-2 (geometric mean 6.1 + 1.4 compared to 2.5 +/- 1.2) . In all foals antibody levels were hardly detectable against both R . equi and EHV-2 until five weeks of age . From the fifth week, antibody levels gradually increased and by the ninth week their reached a titre of 5.5 +/- 1.8 (2.7 +/- 1.2 in the control foals) against R . equi and 5.2 +/- 1.4 against EHV-2 . The favorable clinical results and the low antibody titres in the sera of the vaccinated foals during the first week of life suggest that protection probably was due to repeated vaccination of young foals rather than to vaccination of mares.

Vet Microbiol, 1997 Jun 16, 56(3-4), 193 - 204
Immunoprophylaxis of Rhodococcus equi pneumonia in foals; Becu T et al.; An immunoprophylaxis program for R . equi infection of foals has been established on a number of thoroughbred breeding farms in Argentina over the past 4 years . Nearly 800 mares annually were immunized subcutaneously during the last 2 months of pregnancy with 2-3 doses of a vaccine containing soluble antigens of R . equi, including the virulence associated protein (VapA) and 'equi factors' exoenzymes . The mortality from R . equi pneumonia in the foals from vaccinated dams dropped from an average of 3% in the 5 years before the vaccination program was initiated to an average of 1.2% in the 4 years during which the program was applied (P < 0.02) . On 3 farms, an additional 380 foals of vaccinated dams annually over 3 years also received at 25 days of age 600-1200 ml of hyperimmune plasma from donors immunized with this vaccine, and as well at 4 days of age in foals with poor transfer of R . equi antibodies from their dams . The average foal mortality because of R . equi in the 380 foals annually to which hyperimmune plasma was administered dropped from 5.8% on these 3 farms to 0.2% (P < 0.05) . Active vaccination of foals of unvaccinated mares on an enzootic farm at 20, 30, and 40 days of age did not protect them from mortality due to R . equi pneumonia . Serology was done by complement fixation and an agar gel immunodiffusion (AGID) tests using antigens prepared in the same manner as the vaccine antigens . The immune responses among hyperimmune plasma donors varied considerably as did the responses of vaccinated mares . Of 1117 serum samples with normal post suckling gammaglobulin levels (> 600 mg%) collected at 2 days of age from foals of vaccinated mares, 36% showed a negative or weak positive AGID reaction, while the remainder had positive to strongly positive reactions.

Vet Microbiol, 1997 Jun 16, 56(3-4), 187 - 92
Protective effect against Rhodococcus equi infection in mice of IgG purified from horses vaccinated with virulence associated protein (VapA)-enriched antigens; Fernandez AS et al.; IgG was purified from horses immunized with repeated doses of virulence associated (VapA) enriched antigens extracted with Triton X-114 from the surface of a virulent strain of R . equi . This IgG were administered to mice immunosuppressed by prior treatment with indomethacin . Mice administered the higher dose were completely protected against intraperitoneal infection with R . equi; mice given the lower dose were partially protected . By contrast, mice administered concentrated nonimmune equine IgG were not protected . This study demonstrates that VapA may be an important antigen involved in humoral protective immunity in R . equi infections caused by foal virulent strains.

Vet Microbiol, 1997 Jun 16, 56(3-4), 177 - 85
Immunity to Rhodococcus equi; Hines SA et al.; Rhodococcal pneumonia is an important, life threatening disease of foals and immunosuppressed humans . Increased knowledge of the mechanisms of protective immunity are required in order to develop an effective immunoprophylaxis strategy for horses and immunotherapeutic regiments for people . Both humoral and cellular components of the immune system may be involved in immune clearance of R . equi . The susceptibility of foals less than 4-6 months of age is postulated to reflect waning maternal antibody, and passive transfer of hyperimmune plasma can provide protection on endemic farms . However, effective clearance is likely to require appropriate cellular responses, including the secretion of cytokines . In murine models, both CD4+ and CD8+ T lymphocytes can reduce bacterial counts in the lung . CD4+ cells appear to be both required and sufficient, and IFN-gamma is a primary mediator . Clearance appears to be a type 1 immune response while type 2 responses may lead to a failure to clear and lesion development . It remains to be determined how the cellular immunity experiments reported in mice relate to horses and humans . Likewise, the role of specific R . equi antigens in protective immunity has not been determined.

Vet Microbiol, 1997 Jun 16, 56(3-4), 167 - 76
Epidemiology of Rhodococcus equi infections: a review; Takai S; An overview of epidemiology of R . equi infection in foals is presented, emphasizing the importance of the virulence-associated antigens and plasmids as epidemiological markers . The monoclonal antibody-based colony blot test has been developed to identify rapidly and accurately virulent R . equi . Epidemiological studies conducted during the recent 5 years have revealed that: (1) avirulent R . equi are widespread in the feces of horses and their environment on every farm; (2) the feces of horses and the environment of the horse farms having endemic R . equi infections demonstrated heavy contamination with virulent R . equi, but the farms without the problem did not, thus suggesting that foals bred on a farm with endemic disease are exposed more frequently to virulent R . equi in their environment than those of a farm without the problem; (3) only virulent R . equi are isolated from lesions of naturally infected foals, showing that natural infections in foals are principally by virulent R . equi, but not avirulent organisms; (4) infected foals which constantly shed large quantities of virulent R . equi in their feces are the major source of virulent R . equi, which this may be the mechanism of progressive development of infection on farms with a history of the disease . At present, farms with a potential for endemic infection can be distinguished on the basis of the contamination with virulent R . equi, so regular examination of foals and their environment by virulence markers might be the most practical approach to control R . equi infection on endemic farms.

FEBS Lett, 1997 Jun 9, 409(2), 216 - 20
Highly efficient control of iron-containing nitrile hydratases by stoichiometric amounts of nitric oxide and light; Bonnet D et al.; The reaction of two iron-containing nitrile hydratases (NHase) with NO has been studied: NHase from Rhodococcus sp . R312, which is probably similar to the photosensitive N771 NHase, and the new NHase from Comamonas testosteroni NI1 whose aminoacid sequence is quite different from those of BR312 and N771 NHases . Both enzymes are equally inactivated after addition of stoichiometric amounts of NO added as an anaerobic solution or produced in situ under physiological conditions by a rat brain NO-synthase . Both enzymes are reactivated by photoirradiation, and two cycles of NO inactivation/photoactivation can be performed without significant loss of activity . Both iron-containing NHases have a high affinity for NO, similar to that of methemoglobin.

Appl Biochem Biotechnol, 1997 Jun, 66(3), 281 - 9
Improvement of lysine production by analog-sensitive and auxotroph mutants of the acetylene-utilizing bacterium Gordona bronchialis (Rhodococcus bronchialis); Kyriacou A et al.; 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).

J Clin Microbiol, 1997 Jun, 35(6), 1642 - 4
Prevalence of the virulence-associated gene of Rhodococcus equi in isolates from infected foals; Haites RE et al.; The prevalence of the plasmid-encoded virulence-associated gene (vapA) of Rhodococcus equi, as determined by PCR, was found to be 98% in isolates from 154 foals with pneumonia, confirming the strong association of vapA with virulence . The vapA genes from 60 representative isolates were compared by digestion with the restriction endonuclease HinfI, and no evidence of sequence variation was detected.

Structure, 1997 May 15, 5(5), 691 - 9
Crystal structure of nitrile hydratase reveals a novel iron centre in a novel fold; Huang W et al.; BACKGROUND: Nitrile hydratases are unusual metalloenzymes that catalyze the hydration of nitriles to their corresponding amides . They are used as biocatalysts in acrylamide production, one of the few commercial scale bioprocesses, as well as in environmental remediation for the removal of nitriles from waste streams . Nitrile hydratases are composed of two subunits, alpha and beta, and they contain one iron atom per alphabeta unit . We have determined the crystal structure of photoactivated iron-containing nitrile hydratase from Rhodococcus sp . R312 to 2.65 A resolution as a first step in the elucidation of its catalytic mechanism . RESULTS: The alpha subunit consists of a long N-terminal arm and a C-terminal domain that forms a novel fold . This fold can be described as a four layered structure, alpha-beta-beta-alpha, with unusual connectivities between the beta strands . The beta subunit also contains a long N-terminal extension, a helical domain, and a C-terminal domain that folds into a beta roll . The two subunits form a tight heterodimer that is the functional unit of the enzyme . The active site is located in a cavity at the subunit-subunit interface . The iron centre is formed by residues from the alpha subunit only-three cysteine thiolates and two mainchain amide nitrogen atoms are ligands . CONCLUSIONS: Nitrile hydratases contain a novel iron centre with a structure not previously observed in proteins; it resembles a hybrid of the iron centres of heme and Fe-S proteins . The low-spin electronic configuration presumably results in part from two Fe-amide nitrogen bonds . The structure is consistent with the metal ion having a role as a Lewis acid in the catalytic reaction.

Epidemiol Mikrobiol Imunol, 1997 May, 46(2), 58 - 66
{Rhodococcus equi--a newly recognized opportunistic pathogen in man}; Votava M et al.; The review informs about substantial features of Rhodococcus equi with emphasis on the analysis of 115 as yet published and still expanding reports on the isolation of this zoopathogenic nocardioform actinomycete from man . Microbiological laboratories of human medicine have to learn not only how to identify R . equi but also recognize it as an opportunistic pathogen in particular in persons with the deficient immunity . R . equi is a gram-positive, encapsulated diphtheroid coccobacillus, partially acid fast . It grows well on common media, its colonies being after 48 hours characteristically mucoid, coalescing, irregular and mostly lightly pinkish . Biochemically it is little active, nevertheless it causes typical synergic haemolysis of erythrocytes influenced by staphylococcal beta-toxin . R . equi is found in soil and manure, especially in horse manure . It causes above all granulomatous pneumonia in young foals . In humans, it causes mostly pneumonia and lung abscess, more frequently in persons with immunity deficiency incl . AIDS, less often extrapulmonary abscesses, sepsis and wound infections . The disease are commonly chronic and recurrent . The ability of R . equi to persist in macrophages and destroy them is important in the pathogenesis of infection . In resistance to infection the cell-mediated immunity seems to be of major importance . The port of entry are the lungs, less often the alimentary tract or injured skin . About a third of the persons gives a history of contact with animals, manure or soil . The standard treatment is prolonged administration of a combination of rifampin and erythromycin . The isolation of R . equi is easy and if a laboratory suspects the presence of this microorganism, its identification is not difficult.

Appl Microbiol Biotechnol, 1997 May, 47(5), 583 - 9
Cell-linked and extracellular cholesterol oxidase activities from Rhodococcus erythropolis . Isolation and physiological characterization; Sojo M et al.; Rhodococcus erythropolis cells growing in a cholesterol-free glycerol-containing mineral medium displayed very low levels of a cell-wall-bound cholesterol oxidase activity . Addition of cholesterol induced a marked increase in the synthesis of this enzyme, which reached a maximum within 6 days and was subsequently followed by the appearance of extracellular cholesterol oxidase in the culture broth . Significant levels of induction were only achieved when cholesterol emulsified with Tween 80 . The presence of chloramphenicol at the time of induction completely prevented the emergence of both enzymatic forms, suggesting the requirement of de novo protein synthesis . Upon transfer of cholesterol-growing cultures to fresh medium lacking cholesterol, the extracellular cholesterol oxidase was quickly erased, while the activity of the particulate enzyme decreased sharply . The electrophoretic pattern on native Western blotting as well as on sodium dodecyl sulphate/polyacrylamide gels, together with kinetic data, strongly support the idea that the particulate and extracellular cholesterol oxidases are two different forms of the same enzyme with an estimated molecular mass of 55 kDa.

Appl Environ Microbiol, 1997 May, 63(5), 2062 - 6
Molecular analysis of the Rhodococcus sp . strain H1 her gene and characterization of its product, a heroin esterase, expressed in Escherichia coli; Rathbone DA et al.; The structural gene for heroin esterase was cloned from Rhodococcus sp . strain H1 and expressed in Escherichia coli BL21(DE3) . The purified enzyme was found to be a tetramer with an M(r) of 137,000 and an apparent K(m) of 0.88 mM for 6-acetylmorphine . The G-x-S-x-G motif was observed in the deduced amino acid sequence, suggesting that the enzyme is a serin esterase.

Appl Environ Microbiol, 1997 May, 63(5), 1911 - 6
Thiocarbamate herbicide-inducible nonheme haloperoxidase of Rhodococcus erythropolis NI86/21; De Schrijver A et al.; During biodegradation of thiocarbamate herbicides by Rhodococcus erythropolis NI86/21, a protein with an M(r) of 30,000 is induced (I . Nagy, G . Schoofs, F . Compernolle, P . Proost, J . Vanderleyden, and R.De Mot, J . Bacteriol . 177:676-687, 1995) . Based on N-terminal sequence data for the protein purified by two-dimensional electrophoresis, the corresponding structural gene, thcF, was cloned and sequenced . The deduced protein sequence of ThcF is homologous to those of nonheme haloperoxidases . A particularly high level of sequence identity (72.6%) was observed for the chloroperoxidase from Pseudomonas pyrrocinia . A polyclonal antibody against the latter enzyme cross-reacted with ThcF either produced by the original Rhodococcus cells or overexpressed heterologously in Escherichia coli . In both thiocarbamate-grown Rhodococcus cells and E . coli cells expressing thcF, the haloperoxidase activity of ThcF was demonstrated . The thiocarbamate-inducible R . erythropolis ThcF protein represents the first (nonheme) haloperoxidase to be identified in a nocardioform actinomycete.

Immunopharmacol Immunotoxicol, 1997 May, 19(2), 147 - 64
Pathogenetic role of phagocytic abnormalities in human virus immunodeficiency infection: possible therapeutical approaches . A review; Covelli V et al.; Polymorphonuclear cells (PMN) and monocytes/macrophages (M/M) represent the first defence line against invading microorganisms . Both phagocytic cell functions are precociously compromised in human immunodeficiency virus (HIV)-infected subjects, thus leading to infectious and neurological complications in the late stages of disease . Among intracellular pathogens, emerging bacteria such as Bartonella henselae and Rhodococcus equi can cause peculiar clinical pictures, i.e . the bacillary parenchimal angiomatosis and a classical pyogranulomatous broncopneumonia, respectively . On the other hand, overproduction of proinflammatory cytokines (CKs) and, in particular, tumor necrosis factor-alpha under HIV or lipopolysaccharide stimulation may cause neural damage in terms of demyelination and subsequent development of acquired immunodeficiency syndrome (AIDS) dementia complex . Some therapeutical attempts have been made with colony stimulating factors in order to increase the number and potentiate the function of PMN and M/M . On the other hand, the use of drugs able to reduce exaggerated release of CKs by M/M is suggested in AIDS patients in order to prevent a further aggravation of the clinical condition.

J Clin Microbiol, 1997 Apr, 35(4), 817 - 22
Rapid identification of clinically significant species and taxa of aerobic actinomycetes, including Actinomadura, Gordona, Nocardia, Rhodococcus, Streptomyces, and Tsukamurella isolates, by DNA amplification and restriction endonuclease analysis; Steingrube VA et al.; A previously described PCR-restriction fragment length polymorphism (RFLP) identification schema for Nocardia that used an amplified 439-bp segment (amplicon) of the 65-kDa heat shock protein gene was evaluated for potential use with isolates of all clinically significant aerobic actinomycetes . The study included 28 reference (American Type Culture Collection) strains and 198 clinical isolates belonging to 20 taxonomic groups . Of these 198 isolates, 188 could be differentiated by this PCR-RFLP method . Amplicons from all aerobic actinomycete isolates lacked BstEII recognition sites, thereby distinguishing them from those of mycobacteria that contain one or more such sites . Of 29 restriction endonucleases, MspI plus HinfI produced RFLP patterns that differentiated 16 of the 20 taxa . A single RFLP pattern was observed for 15 of 20 taxa that included 65% of phenotypically clustered isolates . Multiple patterns were seen with Gordona bronchialis, Nocardia asteroides complex type VI, Nocardia otitidiscaviarum, Nocardia transvalensis, and Streptomyces spp . Streptomyces RFLP patterns were the most heterogeneous (five patterns among 19 isolates), but exhibited a unique HinfI fragment of > 320 bp . RFLP patterns that matched those from type strains of Streptomyces albus, Streptomyces griseus, or Streptomyces somaliensis were obtained from 14 of 19 Streptomyces isolates . Only 10 of 28 isolates of N . otitidiscaviarum failed to yield satisfactory amplicons, while only 6 of 188 (3.2%) clinical isolates exhibited patterns that failed to match one of the 21 defined RFLP patterns . These studies extended the feasibility of using PCR-RFLP analysis as a rapid method for the identification of all clinically significant species and taxa of aerobic actinomycetes.

FEMS Immunol Med Microbiol, 1997 Apr, 17(4), 251 - 62
Virulent and avirulent Rhodococcus equi infection in T-cell deficient athymic nude mice: pathologic, bacteriologic and immunologic responses; Madarame H et al.; We investigated the pathologic, bacteriologic and immunologic responses of BALB/c-nu/nu mice (nude mice) and BALB/c mice (euthymic mice) infected intravenously with virulent and avirulent Rhodococcus equi ATCC 33701, and its plasmid-cured derivative ATCC 33701P-, to evaluate the role of T lymphocytes . Adaptive transfer of immune and normal spleen cells into nude mice was also investigated . Nude and euthymic mice were inoculated with 10(6) ATCC 33701 or 10(6) ATCC 33701P- intravenously (i.v.) and killed at 0, 7, 14, 21, 28 and 35 days post-inoculation, except dead cases . In athymic nude mice infected with ATCC 33701, deteriorating systemic inflammatory responses developed during the experimental period and multiplication of the bacteria continued until the end of the experiment . Nude mice developed splenomegaly and multifocal gross hepatic necrosis with some mortality . Splenomegaly was caused by diffuse proliferation of bacteria-laden macrophages and epithelioid cells, and gross hepatic necrosis was caused by the formation of thromboses and granulomatous lesions . Infection of euthymic mice with a sublethal dose of ATCC 33701 resulted in transient granuloma formation in the liver and spleen, production of specific antibodies against the virulent bacteria and gradual elimination thereof . In contrast, infection with ATCC 33701P- produced few lesions after rapid elimination and no antibody production against bacteria in either normal or athymic nude mice . In nude mice given normal and immune spleen cells, histopathological lesions and granulomas formed only in the liver and spleen, in addition to specific antibodies against 15- to 17-kDa antigens . The pathological lesions observed in the nude mice given immune spleen cells were similar to those seen in the mice given normal spleen cells, but they were less severe than those in mice given normal spleen cells . Mice given immune spleen cells showed a significantly higher elevation of antibody production than mice given normal spleen cells . These results suggested that protection against virulent R . equi in mice depends mainly on cell-mediated immune responses, whereas avirulent R . equi in mice are cleared by innate immune responses.

Am J Vet Res, 1997 Apr, 58(4), 356 - 9
Use of Rhodococcus equi virulence-associated protein for immunization of foals against R equi pneumonia; Prescott JF et al.; OBJECTIVE: To evaluate use of the virulence-associated protein of Rhodococcus equi in immunizing foals against R equi pneumonia . ANIMALS: Eight (experimental group) and 6 (controls) mares with their foals . PROCEDURE: Virulence-associated protein extracted from R equi was used to prepare an acetone-precipitated . Triton X-extracted (APTX) antigen . After determination of the efficacy of passive immunization, in untreated foals or in foals given plasma from a horse vaccinated with APTX antigen or from a nonvaccinated horse, a field trial was done to evaluate the efficacy of vaccination of 8 mares, twice with APTX before parturition, and of their foals at ages 3 and 5 weeks; 6 mares and their foals served as unvaccinated controls . All 2-day-old foals were given plasma from local donor horses inoculated with a locally produced bacterin . Serum opsonizing activity produced by vaccination with APTX was determined . Passively immunized foals were challenge exposed with an aerosol of virulent R equi . Foals of the field trial were exposed to enzootic R equi infection . RESULTS: Inoculation with APTX resulted in high IgG antibody liters with opsonizing activity . Passive immunization of foals with plasma from an immunized horse enhanced bacterial clearance from the lungs, compared with that in foals not given plasma or given plasma without APTX antibodies . Vaccination of mares and foals exposed to natural infection resulted in development of R equi pneumonia in 4 of 8 vaccinated foals, but in only 1 of 6 unvaccinated foals . CONCLUSIONS: Vaccination with APTX antigen led to high-titer, opsonizing antibody . Plasma from a vaccinated horse appeared to enhance clearance of R equi from the lungs of foals . Paradoxically, vaccination of mares and their foals with APTX antigen did not protect foals and may have enhanced R equi pneumonia in the foals.

J Bacteriol, 1997 Apr, 179(8), 2772 - 6
Characterization of the genes encoding a receptor-like histidine kinase and a cognate response regulator from a biphenyl/polychlorobiphenyl-degrading bacterium, Rhodococcus sp . strain M5; Labbe D et al.; We report the cloning, sequence, and expression of the bpdS and bpdT genes from Rhodococcus sp . strain M5, which are believed to encode the first two-component signal transduction system in the genus Rhodococcus, which potentially regulates biphenyl/polychlorobiphenyl metabolism in M5 . BpdT has a typical responses regulator sequence (209 amino acids; 23 kDa), whereas BpdS, the predicted histidine kinase component, is an unusually large transmembrane protein (1,576 amino acids; 170 kDa) that contains ATP-binding and leucine-rich repeat motifs and some conserved residues of protein kinases . Expression of bpdST, like that of the bpdC1C2BADE degradative operon, is inducible by biphenyl.

FEMS Microbiol Lett, 1997 Mar 15, 148(2), 175 - 80
Acetylene degradation by new isolates of aerobic bacteria and comparison of acetylene hydratase enzymes; Rosner BM et al.; Aerobic acetylene-degrading bacteria were isolated from soil samples . Two isolates were assigned to the species Rhodococcus opacus, two others to Rhodococcus ruber and Gordona sp . They were compared with known strains of aerobic acetylene-, cyanide-, or nitrile-utilizing bacteria . The acetylene hydratases of R opacus could be measured in cell-free extracts only in the presence of a strong reductant like titanium(III) citrate . Expression of these enzymes was molybdenum-dependent . Acetylene hydratases in cell-free extracts of R ruber and Gordona spp . did not require addition of reductants . No cross-reactivity could be found between cell-free extracts of any of these aerobic isolates and antibodies raised against the acetylene hydratase of the strictly anaerobic fermenting bacterium Pelobacter acetylenicus . These results show that acetylene hydratases are a biochemically heterogeneous group of enzymes.

Gene, 1997 Mar 10, 187(1), 141 - 9
The bphDEF meta-cleavage pathway genes involved in biphenyl/polychlorinated biphenyl degradation are located on a linear plasmid and separated from the initial bphACB genes in Rhodococcus sp . strain RHA1; Masai E et al.; The bphACB genes responsible for the initial oxidation of the aromatic ring of biphenyl/polychlorinated biphenyls (PCB) to meta-cleavage product in Rhodococcus sp . RHA1 have been characterized . We cloned the 6.1 kb EcoRI fragment containing another extradiol dioxygenase gene (etbC) which was induced during the growth on ethylbenzene . The bphD, bphE and bphF encoding 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPD) hydrolase, 2-hydroxypenta-2,4-dienoate hydratase and 4-hydroxy-2-oxovalerate aldolase, respectively, were found downstream of etbC . The deduced amino acid (aa) sequence of RHA1 bphD and bphE had 27-33% and 32-38% identity, respectively, with those of the corresponding genes in Pseudomonas . BphE and BphF are closely related to the corresponding homoprotocatechuate meta-cleavage pathway enzymes of Escherichia coli C . The bphD and bphF were expressed in E . coli and the BphD activity was detected . The etbCphDEF genes were transcribed in biphenyl and ethylbenzene growing cells . Pulsed field gel electrophoresis (PFGE) analysis indicated that RHA1 contains three large linear plasmids . Southern blot analysis indicated that the meta-cleavage pathway for biphenyl/PCB catabolism in RHA1 is directed by the 390 kb plasmid borne bphDEF genes located separately from bphACB gene cluster on the 1100 kb plasmid.

Panminerva Med, 1997 Mar, 39(1), 61 - 3
Rhodococcus equi pneumonia in a patient with occult HIV infection: successful therapy; Volpino P et al.; We report a case of Rhodococcus equi cavitary pneumonia in a 37-year-old patient with occult HIV infection . Because of his good immune status, the patient was given oral erythromycin and rifampin which rapidly resolved the infection . This modality of treatment may be sufficient in HIV-positive selected patients fur the resolution of Rhodococcus equi pneumonia.

Eur J Clin Microbiol Infect Dis, 1997 Mar, 16(3), 241 - 4
Acute mediastinitis due to Rhodococcus equi in a patient with human immunodeficiency virus infection; Casado JL et al.; A patient with human immunodeficiency virus infection developed anterior mediastinitis during antibiotic treatment for empyema due to Rhodococcus equi . This is the first reported case of infection with this organism in this setting . Despite an adequate course of therapy and maintenance treatment with antibiotics to which the isolate of Rhodococcus equi was susceptible in vitro, the patient experienced relapse of the infection into the mediastinum . Clinicians should consider this complication when Rhodococcus equi is present in pleural effusions.

Med Clin North Am, 1997 Mar, 81(2), 319 - 43
Bacterial infections; Kovacs A et al.; Non-opportunistic bacterial infections are an important cause of morbidity and mortality for HIV-infected adults and children . Factors associated with increased risk of these include altered B- and T-cell function; altered phagocytic cell function; skin and mucous membrane defects; and use of indwelling vascular catheters, antibiotics, or cytotoxic agents . The pathogens encountered most frequently are S . aureus, S . pneumoniae, H . influenzae, Salmonella sp., and Pseudomonas aeruginosa . Less commonly encountered organisms include Rhodococcus equi, Listeria monocytogenes, Shigella sp., and Nocardia asteroides, Strategies for prevention as well as diagnosis and treatment of these are discussed.

J Clin Microbiol, 1997 Mar, 35(3), 738 - 40
Restriction enzyme analysis of the virulence plasmids of VapA-positive Rhodococcus equi strains isolated from humans and horses; Nicholson VM et al.; Restriction enzyme digestion patterns of the large virulence plasmids of 8 human and 37 foal isolates of virulence-associated protein (VapA)-positive Rhodococcus equi strains from different sources were compared . Foal isolates came from five continents . Digestion with EcoRI divided these plasmids into three closely related types, and digestion with BamHI divided them into three major types which corresponded to the EcoRI types . The only EcoRI and BamHI type 3 plasmid was from a single foal isolate obtained from Japan . There are thus two major but related virulence plasmids in isolates from foals . Geographic differences were noted, since foal isolates with the EcoRI type 1 plasmid digestion pattern tended to come mostly from the United States, Canada, European countries, India or Zimbabwe and foal isolates with EcoRI type 2 pattern tended to come mostly from Latin American countries . Only 8 of 38 different human isolates, mostly from AIDS patients, were VapA positive, in contrast to 37 of 42 foal isolates . VapA-positive isolates from humans possessed virulence plasmids of either EcoRI type 1 or EcoRI type 2 . These results confirm that only a small proportion of human patients with R . equi infections acquire foal virulent R . equi.

Berl Munch Tierarztl Wochenschr, 1997 Feb, 110(2), 54 - 9
{Characterization of Rhodococcus equi isolates from horse and man}; Fuhrmann C et al.; In the present investigation 19 and 22 R . equi-cultures isolated from diseased horses and humans, respectively, could be correctly identified by their morphological, biochemical and serological properties . The rod-coccus life cycle appeared to be a common feature of almost all cultures investigated . The cultures were typeable with the typing system described by Prescott (1981) . The predominant serotypes among the R . equi-isolates belonged to serotypes 1 and 2 . Among the R . equi-isolates from horses haemagglutination-positive cultures were mainly found among isolates of serotype 1, those of serotype 2 were haemagglutination-negative . The R . equi-cultures isolated from humans showed no relation between serotype and haemagglutinating properties . Determination of antibiotic susceptibility revealed that all cultures were susceptible to erythromycin, gentamicin, imipenem, minocycline, neomycin, rifampicin, streptomycin and vancomycin, 71% and 75% were resistant or at least moderate susceptible to tetracycline and penicillin G, respectively . Almost all cultures were resistant to ceftazidime and most cultures were susceptible to cefotaxime . The cultures could be further characterized by restriction endonuclease digestion and pulsed field gel electrophoresis of their chromosomal DNA . After digestion with the restriction enzyme AsnI the resulting DNA-profile allowed a strain-specific characterization.

Gene, 1997 Jan 31, 185(1), 49 - 54
Classification of catechol 1,2-dioxygenase family: sequence analysis of a gene for the catechol 1,2-dioxygenase showing high specificity for methylcatechols from Gram+ aniline-assimilating Rhodococcus erythropolis AN-13; Murakami S et al.; Gram+ aniline-assimilating Rhodococcus erythropolis AN-13 (AN-13) produces catechol 1,2-dioxygenase (C12O) showing high enzymatic activities for 3- and 4-methylcatechols {Aoki et al . (1984) Agric . Biol . Chem . 48, 2087-2095} . A 3.0 kb Sau3AI fragment carrying a gene encoding C12O(catA) was cloned by selection of transformants showing C12O activity from a gene library of AN-13 . Furthermore, we specified a 1.6 kb SalI fragment containing catA from the Sau3AI fragment by subcloning . Sequence analysis revealed that the 1.6 kb SalI fragment carried a 855 bp open reading frame (ORF) encoding the entire AN-13 catA, preceded by a potential ribosome binding site (RBS) . From comparison of the deduced amino acid (aa) sequence of C12O from AN-13 with other C12O reported previously, it was found that the AN-13 enzyme shares 56.0% aa sequence identity with C12o from Arthrobacter sp . mA3 (mA3) {Eck and Belter (1991) Gene 123, 87-92} compared with less than 36.4% aa sequence identities with others . In conclusion, we classified all C12O including the AN-13 enzyme into three subfamilies on the basis of similarity of aa sequences, numbers of aa residues, and substrate specificity.

Biochem Biophys Res Commun, 1997 Jan 3, 230(1), 73 - 5
A flavin reductase stimulates DszA and DszC proteins of Rhodococcus erythropolis IGTS8 in vitro; Xi L et al.; Rhodococcus erythropolis IGTS8 is a gram positive bacterium, which can catabolize dibenzothiophene to 2-hydroxybiphenyl and inorganic sulfur without the cleavage of carbon-carbon bonds . Three structural genes, dszA, dszB, and dszC, have been cloned and shown to be necessary for this phenotype . Here, we demonstrate that a FMN:NADPH oxidoreductase from Vibrio harveyi complements activities of purified DszA and DszC proteins . Furthermore, we propose that DszA and DszC are oxygenase units that do not use NAD(P)H directly, but instead use FMNH2 from a FMN:NADPH oxidoreductase for oxygenation.

FEBS Lett, 1997 Jan 2, 400(1), 83 - 90
Subunit topology of the Rhodococcus proteasome; Zuhl F et al.; The 20S proteasome, isolated from the nocardioform actinomycete Rhodococcus erythropolis strain NI86/21, is built from two alpha-type and two beta-type subunits . In order to probe the subunit topology, we have set up an expression system which allows coexpression of the genes encoding the alpha- and beta-subunits in all possible combinations . The four respective constructs obtained yielded fully assembled and proteolytically active proteasomes . Biochemical, kinetic and electron microscopy analysis allow us to rule out several of the models which were originally envisaged for the subunit topology of the Rhodococcus proteasome . The experiments further indicate that the assembly pathways of the Rhodococcus and of the Thermoplasma proteasome differ in some important details.

DNA Seq, 1997, 7(3-4), 225 - 8
Further sequence analysis of the DNA regions with the Rhodococcus 20S proteasome structural genes reveals extensive homology with Mycobacterium leprae; Nagy I et al.; The sequence of the respective DNA regions downstream of the 20S proteasome structural genes prcB1A1 (6 kb) and prcB2A2 (3.3 kb) of Rhodococcus erythropolis NI86/21 were determined . A highly conserved gene organization was observed between the two clusters which differed significantly in G + C content (68.8% versus 62.6%) . Several ORFs were homologues of putative genes previously identified by genomic sequencing of the equivalent DNA in the related nocardioform actinomycete, Mycobacterium leprae, and thought to be specific for this pathogen . Three ORFs (ORF8(1), ORF8(2), ORF12{1}) without a counterpart in M . leprae were found . No significant homology to known sequences including proteasome-related gene products was detected, except for ORF9(1) and ORF9(2) which display a high level of sequence identity with a partially sequenced ORF in Streptomyces chrysomallus . These downstream ORFs also show a significant level of sequence homology with the ORF6(1) and ORF6(2) which are located upstream of the proteasome structural genes in the respective clusters.

Can J Microbiol, 1997 Jan, 43(1), 17 - 22
Bacterial degradation of emulsified crude oil and the effect of various surfactants; Bruheim P et al.; A Rhodococcus sp . 094 bacterium was tested for its ability to oxidize alkanes in crude oil emulsified by nonionic chemical and biological surfactants . Oxidation rates were measured in a 3-h period by Warburg respirometry . 14CO2 recovery was measured from the {1-14C}hexadecane spiked crude oil . Response to emulsified oil depended on the physiological state of the bacteria (i.e., cells harvested in the exponential and stationary growth phases) were tested . Oxidation rates by cells in the exponential growth phase were negatively affected by surfactant amendment . Oxidation rates by cells in the stationary growth phase were in some cases stimulated by surfactants . The stimulatory effect depended on both the chemical structure and the physicochemical properties (i.e., hydrophilic-lipophilic balance (HLB)) of the surfactants . Surfactants with intermediate HLB values (8-12) gave the best results . Neither the biosurfactants nor the commercial oil-spill dispersants tested had any significant stimulatory effect.

Eur J Radiol, 1997 Jan, 24(1), 66 - 70
Radiologic features of Rhodococcus equi pneumonia in AIDS; Muntaner L et al.; This report outlines the radiological features observed in three cases of Rhodococcus equi (R . equi) pneumonia in AIDS (acquired immunodeficiency syndrome) and reviews another 45 radiological reports published of this emerging opportunistic pneumonia in Human Immunodeficiency Virus (HIV) infected patients . The clinical signs in our three patients consisted in a subacute onset of respiratory symptoms and fever . A low lymphocyte count (< 200 cells/mm3), pulmonary infiltrates, and pleural effusion was present in all three cases . Cavitary pneumonia was observed in two patients, and pericardial effusion in another . In this series CD4 lymphocyte count < 200/mm3 was seen in 29 of the 48 patients (60.4%) . All 48 patients had abnormal findings on chest radiographs . Abnormalities involved the upper lobes in 26 of the 48 patients (55%) . Cavitation was reported in 37 of the 48 cases (77%) . R . equi pneumonia may not be as the paucity of case reports suggest . Consequently, a cavitary pneumonia in HIV infected patients with a low CD4 lymphocyte count (< 200 mm3) with a subacute onset, an upper lobe predilection, and/or a poor response to conventional antibiotic therapy should be considered as suspect of R . equi infection.

Eur Respir J, 1997 Jan, 10(1), 248 - 50
Chronic pneumonia caused by Rhodococcus equi in a patient without impaired immunity; Linares MJ et al.; A 48 year old, human immunodeficiency virus (HIV)-negative, immunocompetent male patient had a chronic progressive pulmonary infiltrate, without radiological cavitation, in the middle lobe of the right lung produced by Rhodococcus equi . He reported direct contact with a diseased dog . The patient was diagnosed by thoracotomy and treated by lobectomy . After 16 months of follow-up, the patient was asymptomatic and had neither recurrence nor immunological disturbances.

Microbiology, 1997 Jan, 143 ( Pt 1), 109 - 15
The plasmid-located haloalkane dehalogenase gene from Rhodococcus rhodochrous NCIMB 13064; Kulakova AN et al.; The haloalkane dehalogenase (dhaA) gene from Rhodococcus rhodochrous NCIMB 13064 was cloned and sequenced . Its comparison with the previously studied dhlA gene from Xanthobacter autotrophicus GJ10 did not show homology . However, the amino acid sequences of the products of these genes showed approximately 30% identity and several of the catalytic amino acid residues were conserved in the NCIMB 13,064 dehalogenase . A high level of dhaA expression was demonstrated in Escherichia coli cells and this gene was shown to encode a dehalogenase with the activity against chloroalkanes of chain length C3-C10 . Also, some dehalogenase activity against 1,2-dichloroethane encoded by the cloned dhaA gene was detected . The analysis of NCIMB 13,064 derivatives lacking dehalogenase activity showed that the dhaA gene was located on the 100 kbp pRTL1 plasmid . It was also found that reversible rearrangements of DNA in the dhaA region may be responsible for the control of expression of haloalkane dehalogenase in R . rhodochrous NCIMB 13064 . A number of repeated and inverted sequences which may cause genetic instability at the locus were found in the haloalkane dehalogenase gene region.

Radiographics, 1997 Jan-Feb, 17(1), 47 - 58; discussion 59-61
Interpretation of chest radiographs in AIDS patients: usefulness of CD4 lymphocyte counts; Shah RM et al.; Specific infections and neoplasms that are complications of acquired immunodeficiency syndrome (AIDS) occur within various CD4 lymphocyte count ranges . Knowledge of how these counts correlate with radiographic appearances of these entities can limit the differential diagnosis because certain conditions are uncommon above a specific count . In patients with CD4 lymphocyte counts above 200 cells/mm3 and radiographic findings of cavitary and noncavitary consolidation, bacterial pneumonia and Mycobacterium tuberculosis are the major diagnostic considerations . As the CD4 lymphocyte count falls, these infections are still common; however, cavitation is seen less frequently with Mycobacterium tuberculosis, and unusual bacterial infections, including those caused by Rhodococcus equi and Nocardia asteroides, should be considered . In patients with counts below 200 cells/mm3, Pneumocystis carinii pneumonia is the most common infection, usually manifesting radiographically as a reticular interstitial pattern . At CD4 lymphocyte counts of 50-200 cells/mm3, disseminated fungal infection and Kaposi sarcoma become prevalent . In patients with advanced AIDS and counts below 50 cells/mm3, radiographic nodular or reticular patterns may indicate AIDS-related lymphoma and cytomegalovirus and Mycobacterium avium-intracellulare infections . When CD4 lymphocyte counts are applied to interpretation of chest radiographs in AIDS patients, the working differential diagnosis of a radiographic pattern can be tailored to the clinical situation of a given patient.

J Bacteriol, 1997 Jan, 179(2), 409 - 16
Cloning, analysis, and overexpression of the gene encoding isobutylamine N-hydroxylase from the valanimycin producer, Streptomyces viridifaciens; Parry RJ et al.; The flavoprotein isobutylamine N-hydroxylase (IBAH) catalyzes the oxidation of isobutylamine to isobutylhydroxylamine, a key step in the biosynthesis of the azoxy antibiotic valanimycin . By using oligonucleotide primers designed from peptide sequence information derived from native IBAH, a fragment of the gene (vlmH) encoding IBAH was amplified by PCR from a genomic library of the valanimycin-producing organism, Streptomyces viridifaciens MG456-hF10 . The gene fragment was then employed as a probe to clone the entire vlmH gene from an S . viridifaciens genomic library . Overexpression of the vlmH gene in Escherichia coli gave a soluble protein that was purified to homogeneity . The purified protein exhibited the catalytic activity expected for IBAH . The deduced amino acid sequence of IBAH exhibited the greatest similarity to the Sox/DszC protein from Rhodococcus sp . strain IGT38, a flavoprotein involved in the oxidation of dibenzothiophene to the corresponding sulfone . Significant similarities were also found between the amino acid sequence of IBAH and those of the acyl coenzyme A dehydrogenases.

J Bacteriol, 1997 Jan, 179(2), 370 - 81
Characterization of catechol catabolic genes from Rhodococcus erythropolis 1CP; Eulberg D et al.; The biochemical characterization of the muconate and the chloromuconate cycloisomerases of the chlorophenol-utilizing Rhodococcus erythropolis strain 1CP previously indicated that efficient chloromuconate conversion among the gram-positive bacteria might have evolved independently of that among gram-negative bacteria . Based on sequences of the N terminus and of tryptic peptides of the muconate cycloisomerase, a fragment of the corresponding gene has now been amplified and used as a probe for the cloning of catechol catabolic genes from R . erythropolis . The clone thus obtained expressed catechol 1,2-dioxygenase, muconate cycloisomerase, and muconolactone isomerase activities . Sequencing of the insert on the recombinant plasmid pRER1 revealed that the genes are transcribed in the order catA catB catC . Open reading frames downstream of catC may have a function in carbohydrate metabolism . The predicted protein sequence of the catechol 1,2-dioxygenase was identical to the one from Arthrobacter sp . strain mA3 in 59% of the positions . The chlorocatechol 1,2-dioxygenases and the chloromuconate cycloisomerases of gram-negative bacteria appear to be more closely related to the catechol 1,2-dioxygenases and muconate cycloisomerases of the gram-positive strains than to the corresponding enzymes of gram-negative bacteria.

Antimicrob Agents Chemother, 1997 Jan, 41(1), 218 - 21
Monooxygenase-like sequence of a Rhodococcus equi gene conferring increased resistance to rifampin by inactivating this antibiotic; Andersen SJ et al.; A DNA clone from Rhodococcus equi conferring low-level rifampin resistance through the ability to inactivate this antibiotic via its decomposition was identified . The iri (inactivation of rifampin) gene consisted of an open reading frame of 1,437 bp encoding a 479-amino-acid sequence strongly resembling those of monooxygenases acting upon phenolic compounds or involved in polyketide antibiotic synthesis . When expressed in Escherichia coli, the gene conferred resistance to a > 50-micrograms/ml concentration of the drug.

J Clin Microbiol, 1997 Jan, 35(1), 79 - 85
Routine use of PCR-restriction fragment length polymorphism analysis for identification of mycobacteria growing in liquid media; Taylor TB et al.; A PCR-restriction fragment length polymorphism (PCR-RFLP) procedure capable of rapidly identifying 28 species of clinically encountered mycobacteria was evaluated for use in the routine identification of acid-fast isolates growing in BACTEC 12B and 13A liquid media . PCR-RFLP identified 100 of 103 acid-fast isolates recovered from 610 patient specimens submitted for culture during the study . The three isolates unidentifiable by PCR-RFLP produced restriction patterns not included in the PCR-RFLP algorithm and could therefore not be assigned to a species . These isolates were characterized by their morphologic and biochemical characteristics . Two of the isolates were identified as M . terrae complex and M . gordonae . The third isolate could not be definitively identified and could only be characterized as a Mycobacterium sp . most closely resembling M . chelonae . PCR-RFLP identifications agreed with the conventional identifications for 96 of the 100 isolates identified by PCR-RFLP . Subsequent identification of the four discordant isolates by gas chromatography analysis supported the PCR-RFLP identification of each isolate . Amplification products were also obtained from isolates of Streptococcus albus and Rhodococcus equi recovered from patient specimens; however, the restriction patterns of these nonmycobacterial species did not resemble the patterns of any mycobacterial species included in the PCR-RFLP algorithm . PCR-RFLP seems to be a reliable procedure for the routine identification of mycobacteria and has the potential for providing identifications of mycobacterial isolates which are more accurate than conventional identification techniques based on morphologic and biochemical characteristics.

Biochemistry, 1996 Dec 24, 35(51), 16777 - 81
Resonance Raman evidence that photodissociation of nitric oxide from the non-heme iron center activates nitrile hydratase from Rhodococcus sp . N-771; Noguchi T et al.; Nitrile hydratase (NHase) from Rhodococcus sp . N-771, which contains a non-heme iron center in the catalytic site, has been known to be activated by light illumination . Recently, endogenous nitric oxide (NO) was found in this enzyme by FTIR spectroscopy {Noguchi et al . (1995) FEBS Lett . 358, 9-12} . In order to directly detect the bonding between NO and the iron atom and the reaction of NO upon photoactivation, resonance Raman spectra of the NHase were measured with 413 nm excitation at 85 K . Two prominent bands at 592 and 570 cm-1 were observed in the inactive from, and both of them were completely lost upon photoactivation . Upon subsequent introduction of 15NO, the active NHase was converted to the inactive form again and the above two bands were restored with downshifts by 10 and 12 cm-1, respectively . Also, the excitation profiles of these bands in the 350-500 nm region mostly followed the absorption spectrum arising from the iron center . From these isotopic shifts and the excitation profiles, the two Raman bands were assigned to the Fe-NO stretching and bending vibrations that are probably coupled with each other . The results provided solid evidence that NO is bound to the non-heme iron in the inactive NHase and its photodissociation activates the enzyme.

Gene, 1996 Dec 5, 182(1-2), 215 - 8
Amide metabolism: a putative ABC transporter in Rhodococcus sp . R312; Chebrou H et al.; The DNA sequence has been determined upstream of the amiE structural gene in the amidase operon of Rhodococcus sp . R312 and a new ORF (amiS2) identified . The amiS2 gene encodes a potential 206 amino acid (aa) protein containing a high proportion of hydrophobic residues . The AmiS2 protein possesses high homology to the ORFP3, amiS and ureI gene products from the Mycobacterium smegmatis (Ms) acetamidase operon, Pseudomonas aeruginosa (Pa) amidase operon and Helicobacter pylori (Hp) urease operon, respectively . Hydropathic analysis and secondary structure prediction of AmiS2 suggested the presence of seven potential transmembrane (TM) alpha-helices . Sequence analysis of the amiB2 gene, located downstream of the Rhodococcus sp . R312 amiE gene, showed that it encoded a 351-aa protein containing a potential ATP-binding motif . AmiB2 showed significant homology with the ATP-binding subunit of the bacterial Clp protease and high homology with the amiB product located within the Pa amidase operon . AmiB2 and AmiS2 appear to be two components of a recently identified novel family of ABC transporters (Wilson et al., 1995) and might be responsible for the adsorption of amidase substrates or release of their hydrolysis products.

FEMS Microbiol Lett, 1996 Dec 1, 145(2), 227 - 31
Isolation of Rhodococcus rhodochrous NCIMB13064 derivatives with new biodegradative abilities; Kulakova AN et al.; Rhodococcus rhodochrous NCIMB13064 can dehalogenate and utilise a number of halogenated aliphatic compounds as sole carbon and energy source . Mutants of NCIMB13064 can be easily isolated with an enlarged range of 1-chloroalkane utilising ability . Dehalogenation of 1-chlorononane, 1-chlorodecane and short-chain 1-chloroalkanes (C3-C8) is encoded by the same plasmid pRTL1 . However, a different genetic element(s) is required for the dehalogenation of 3-chloropropionic acid . Two derivatives (P200 and P400) of R . rhodochrous NCIMB13064 were isolated which had acquired the ability to utilise naphthalene as sole carbon and energy source . Both strains lost the ability to utilise short-chain 1-chloroalkanes and underwent some rearrangements associated with pRTL1 plasmid.

Diagn Cytopathol, 1996 Nov, 15(4), 325 - 8
Cytologic features of pulmonary malakoplakia related to Rhodococcus equi in an immunocompromised host; van Hoeven KH et al.; Cytologic features are described in bronchial brushings of a large cavitary lung mass from an immunosuppressed patient who had undergone liver transplantation . Scattered histiocytes with abundant eosinophilic, vacuolated cytoplasm were noted in a background of bronchial cells . Within approximately one fourth of the histiocytes, targetoid intracytoplasmic inclusions were present, consistent with Michaelis-Gutmann bodies . They were diffusely positive by histo-chemical staining with von Kossa, Gomori methenamine silver, and periodic acid-Schiff stains, and focally positive with Prussian blue stain . Cultures of the abscess yielded Rhodococcus equi . The characteristic microscopic features of pulmonary malakoplakia can be discerned in bronchial brushings, and should be sought, particularly in immunocompromised patients.

Microbiology, 1996 Nov, 142 ( Pt 11), 3241 - 51
Studies on the isopropylbenzene 2,3-dioxygenase and the 3-isopropylcatechol 2,3-dioxygenase genes encoded by the linear plasmid of Rhodococcus erythropolis BD2; Kesseler M et al.; The enzymes responsible for the degradation of isopropylbenzene (IPB) and co-oxidation of trichloroethene (TCE) by Rhodococcus erythropolis BD2 are encoded by the linear plasmid pBD2 . Fragments containing IPB catabolic genes were cloned from pBD2 and the nucleotide sequence was determined . By means of database searches and expression of the cloned genes in recombinant strains, we identified five clustered genes, ipbA1A2A3A4C, which encode the three components of the IPB 2,3-dioxygenase system, reductaseIPB (ipbA4), ferredoxinIPB (ipbA3) and the two subunits of the terminal dioxygenase (ipbA1A2), as well as the 3-isopropylcatechol (IPC) 2,3-dioxygenase (ipbC) . The protein sequences deduced from the ipbA1A2A3A4C gene cluster exhibited significant homology with the corresponding proteins of analogous degradative pathways in Gram-negative and Gram-positive bacteria, but the gene order differed from most of them . IPB 2,3-dioxygenase and 3-IPC 2,3-dioxygenase could both be expressed in Escherichia coli, but the IPB 2,3-dioxygenase activities were too low to be detected by polarographic and TCE degradative means . However, inhibitor studies with the R . erythropolis BD2 wild-type are in accordance with the involvement of the IPB 2,3-dioxygenase in TCE oxidation.

J Bacteriol, 1996 Nov, 178(22), 6409 - 18
Genetic analysis of the dsz promoter and associated regulatory regions of Rhodococcus erythropolis IGTS8; Li MZ et al.; The dsz gene cluster of Rhodococcus erythropolis IGTS8 comprises three genes, dszA, dszB, and dszC, whose products are involved in the conversion of dibenzothiophene (DBT) to 2-hydroxybiphenyl and sulfite . This organism can use DBT as the sole sulfur source but not as a carbon source . Dsz activity is repressed by methionine, cysteine, Casamino Acids, and sulfate but not by DBT or dimethyl sulfoxide . We cloned 385 bp of the DNA immediately 5' to dszA in front of the reporter gene lacZ of Escherichia coli . We showed that this region contains a Rhodococcus promoter and at least three dsz regulatory regions . After hydrazine mutagenesis of this DNA, colonies that were able to express beta-galactosidase in the presence of Casamino Acids were isolated . Sequencing of these mutants revealed two possible regulatory regions . One is at -263 to -244, and the other is at -93 to -38, where -1 is the base preceding the A of the initiation codon ATG of dszA . An S1 nuclease protection assay showed that the start of the dsz promoter is the G at -46 and that transcription is repressed by sulfate and cysteine but not by dimethyl sulfoxide . The promoter encompasses a region of potential diad symmetry that may contain an operator . Immediately upstream of the promoter is a protein-binding domain between -146 and -121 . Deletion of this region did not affect repression, but promoter activity appeared to be reduced by threefold . Thus, it could be an activator binding site or an enhancer region.

Bone Marrow Transplant, 1996 Oct, 18(4), 813 - 5
Allogeneic bone marrow transplantation can restore CD4+ T-lymphocyte count and immune function in idiopathic CD4+ T-lymphocytopenia; Petersen EJ et al.; CD4+ T-lymphocytopenia in the absence of HIV infection is a heterogeneous disorder of unknown cause . Here we report a patient with idiopathic CD4+ T-lymphocytopenia, presenting with an opportunistic Rhodococcus equi infection . When aplastic anemia developed subsequently, allogeneic bone marrow transplantation was performed . Complete restoration of immune function was observed . We conclude that allogeneic bone marrow transplantation presents a potentially curative therapy for CD4+ T-lymphocytopenia.

Epidemiol Infect, 1996 Oct, 117(2), 393 - 400
Genetic analysis of Clavibacter toxicus, the agent of annual ryegrass toxicity; Johnston MS et al.; Multilocus enzyme electrophoresis was used to examine the relatedness of 52 isolates of Clavibacter toxicus, the agent of annual ryegrass toxicity . These included 37 Western Australian (WA) field isolates sampled in 3 distinct locations over a 2-year period, and 15 isolates sampled from 6 different host plant species in 3 states in Australia over approximately 8 years . Seventeen reference strains for the related genera Curtobacterium, Rhodococcus and Arthrobacter were examined for comparison . The 69 isolates were divided into 29 electrophoretic types (ETs), separated by genetic distances of 0.06 to 0.81 . The C . toxicus isolates fell into 12 ETs, 11 of which formed a tightly clustered group separated by a genetic distance of 0.23 or less . Thirty-one of the WA field isolates of C . toxicus fell into a single ET, and four into another ET . Clavibacter toxicus therefore formed a closely related group which was genetically distinct from the other plant pathogenic species, and a dominant widely disseminated strain of the species was identified in WA.

J Bacteriol, 1996 Oct, 178(19), 5699 - 705
Gene overexpression, purification, and identification of a desulfurization enzyme from Rhodococcus sp . strain IGTS8 as a sulfide/sulfoxide monooxygenase; Lei B et al.; The oxidation of dibenzothiophene to dibenzothiophene sulfone has been linked to the enzyme encoded by the sox/dszC gene from Rhodococcus sp . strain IGTS8 (S . A . Denome, C . Oldfield, L . J . Nash, and K . D . Young, J . Bacteriol . 176:6707-6717, 1994; C . S . Piddington, B . R . Kovacevich, and J . Rambosek, Appl . Environ . Microbiol . 61:468-475, 1995) . However, this enzyme has not been characterized, and the type of its catalytic activity remains unclassified . In this work, the sox/dszC gene was overexpressed in Escherichia coli, a procedure for the purification of the expressed enzyme was developed, and the properties of and the reactions catalyzed by the purified enzyme were characterized . This enzyme binds one flavin mononucleotide (Kd, 7 micrometers) or reduced flavin mononucleotide (FMNH2) (Kd < 10(-8) M) per 90,200-Da homodimer, and FMNH2 is an essential cosubstrate for its activity . Patterns of product formation were examined under different FMNH2 availabilities, and results indicate that this enzyme catalyzes a stepwise conversion of dibenzothiophene to the corresponding sulfoxide and subsequently to the sulfone . On the basis of isotope labeling patterns with H2(18)O and 18O2, dibenzothiophene sulfoxide and sulfone obtained their oxygen atom(s) from molecular oxygen rather than water in their formation from dibenzothiophene . The enzyme also utilizes benzyl sulfide and benzyl sulfoxide as substrates . Hence, it is identified as a sulfide/sulfoxide monooxygenase . This monooxygenase is similar to the microsomal flavin-containing monooxygenase but is unique among microbial flavomonooxygenases in its ability to catalyze two consecutive monooxygenation reactions.

Zentralbl Bakteriol, 1996 Sep, 285(1), 11 - 9
A chemotaxonomic study of the lipoglycans of Rhodococcus rhodnii N445 (NCIMB 11279); Flaherty C et al.; Rhodococcus rhodnii N445 was investigated for the presence of macroamphiphilic lipoglycan . Purification of a hot phenol-water extract by hydrophobic interaction chromatography allowed the resolution of three lipoglycan fractions . The two main preparations contained lipoglycans with carbohydrate compositions consistent with the presence of lipoarabinomannan and lipomannan, whilst the minor fraction appeared to contain a mixture of these two lipoglycans . The fatty acid composition of the lipoglycans resembled that of the whole cells except that the relative proportion of unsaturated fatty acids was decreased . Although lipoarabinomannan and structurally-related lipomannan lipoglycans from representatives of the genus Mycobacterium have been extensively studied, this is the first report of the lipoglycan composition of a representative of the genus Rhodococcus as presently defined . These findings provide further chemotaxonomic evidence that lipoarabinomannan-type lipoglycans are widely distributed throughout the mycolic acid-containing actinomycetes.

J Biochem (Tokyo), 1996 Sep, 120(3), 663 - 70
Relationship between induction of macrophage chemotactic factors and formation of granulomas caused by mycoloyl glycolipids from Rhodococcus ruber (Nocardia rubra); Matsunaga I et al.; Mycoloyl glycolipids cause granulomas in the lungs, liver, and spleen of mice, but the mechanism is not fully understood . To understand the role of macrophage chemotactic factors (MCFs) in granuloma formation, we prepared various mycoloyl glycolipids with different carbohydrate moieties: trehalose dimycolate (TDM), glucose mycolate (GM), mannose mycolate (MM), and fructose mycolate (FM) from Rhodococcus ruber, and examined the relationship between their MCF induction in peritoneal macrophages and the extent of granuloma formation . The molecular mass of each glycolipid was confirmed by fast-atom-bombardment mass-spectrometry . TDM or GM caused granulomas in the lungs, spleen, and liver of ICR mice, but MM and FM did not . The culture supernatant of peritoneal macrophages stimulated with TDM or GM increased macrophage migration, whereas MM and FM had no chemotactic activity . The activity of interleukin-1 (IL-1) in the supernatant was increased equally by each glycolipid and was therefore not related to chemotaxis . Tumor necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were not detected in the four supernatants . The TDM-induced MCF was heat-stable, trypsin-labile, and undialyzable . Furthermore, we separated two MCF active fractions from the supernatant of TDM-stimulated macrophages by gel filtration . These factors acted on macrophages but not on neutrophils . Our results suggested that macrophages recognize the sugar moieties of mycoloyl glycolipids and may, in response, generate a MCF that may play an important role in the macrophage or monocyte recruitment which is essential prior to granuloma formation.

Equine Vet J, 1996 Sep, 28(5), 344 - 9
Use of a virulence-associated protein based enzyme-linked immunosorbent assay for Rhodococcus equi serology in horses; Prescott JF et al.; An enzyme-linked immunosorbent assay (ELISA) was developed against Rhodococcus equi using Triton X-114 detergent extracted whole cell material, in which the virulence associated protein (VapA) predominated . Enzymelinked immunosorbent assay titres corresponded to antibody reacting with VapA on Western blots . There was considerable variation in antibody titres of nonimmunised mares and in the time when the colostrally derived antibody of their foals had declined to low or undetectable titres . In general, antibodies in foals declined to their lowest levels at age 4-8 weeks . Seroconversion occurred in foals age 8-10 weeks, but the precise time depended on maternal titre and the month in which the foal was born . Foals reaching age 8 weeks in late summer showed more marked seroconversion than foals born earlier . The ELISA was used to follow the response to immunisation with the same Triton X-114 extracted material . Six mares immunised before parturition with the antigen in aluminium hydroxide adjuvant developed high titres, up to > 102,400 and transferred them to their foals through colostrum . Their foals responded to immunisation with 0.5-1.0 mg antigen 3, 5, 7 and 9 weeks after birth . Antibody titres following immunisation with similar dosage reached up to > 102,400 in a separate group of foals of nonimmunised mares . Nonvaccinated control foals seroconverted at age 6-8 weeks . The VapA based ELISA is useful to follow the course of natural infection with R . equi or immunisation with VapA based antigen.

Lett Appl Microbiol, 1996 Aug, 23(2), 72 - 4
Identification of Rhodococcus equi using the polymerase chain reaction; Bell KS et al.; Two regions in the gene coding for 16S rRNA in Rhodococcus equi were selected as species-specific primer sequences for the polymerase chain reaction (PCR) . PCR using these primers was tested against 10 strains of R . equi (including the type strain) and gave positive results for all but was negative for all other tested species of Rhodococcus; representatives of the most closely related genera and a number of other bacterial species . This method could therefore be used to identify this species which can infect the lungs or other organs of horses, pigs, humans and other animals.

J Bacteriol, 1996 Aug, 178(16), 4894 - 900
Atrazine chlorohydrolase from Pseudomonas sp . strain ADP: gene sequence, enzyme purification, and protein characterization; de Souza ML et al.; Pseudomonas sp . strain ADP metabolizes atrazine to carbon dioxide and ammonia via the intermediate hydroxyatrazine . The genetic potential to produce hydroxyatrazine was previously attributed to a 1.9-kb AvaI DNA fragment from strain ADP (M . L . de Souza, L . P . Wackett, K . L . Boundy-Mills, R . T . Mandelbaum, and M . J . Sadowsky, Appl . Environ . Microbiol . 61:3373-3378, 1995) . In this study, sequence analysis of the 1.9-kb AvaI fragment indicated that a single open reading frame, atzA, encoded an activity transforming atrazine to hydroxyatrazine . The open reading frame for the chlorohydrolase was determined by sequencing to be 1,419 nucleotides and encodes a 473-amino-acid protein with a predicted subunit molecular weight of 52,421 . The deduced amino acid sequence matched the first 10 amino acids determined by protein microsequencing . The protein AtzA was purified to homogeneity by ammonium sulfate precipitation and anion-exchange chromatography . The subunit and holoenzyme molecular weights were 60,000 and 245,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography, respectively . The purified enzyme in H2(18)O yielded {18O}hydroxyatrazine, indicating that AtzA is a chlorohydrolase and not an oxygenase . The most related protein sequence in GenBank was that of TrzA, 41% identity, from Rhodococcus corallinus NRRL B-15444R . TrzA catalyzes the deamination of melamine and the dechlorination of deethylatrazine and desisopropylatrazine but is not active with atrazine . AtzA catalyzes the dechlorination of atrazine, simazine, and desethylatrazine but is not active with melamine, terbutylazine, or desethyldesisopropylatrazine . Our results indicate that AtzA is a novel atrazine-dechlorinating enzyme with fairly restricted substrate specificity and contributes to the microbial hydrolysis of atrazine to hydroxyatrazine in soils and groundwater.

Appl Environ Microbiol, 1996 Aug, 62(8), 2940 - 6
Identification of an alternative 2,3-dihydroxybiphenyl 1,2-dioxygenase in Rhodococcus sp . strain RHA1 and cloning of the gene; Hauschild JE et al.; Gram-positive Rhodococcus sp . strain RHA1 possesses strong polychlorinated biphenyl-degrading capabilities . An RHA1 bphC gene mutant, strain RDC1, had been previously constructed (E . Masai, A . Yamada, J . M . Healy, T . Hatta, K . Kimbara, M . Fukuda, and K . Yano, Appl . Environ . Microbiol . 61:2079-2085, 1995) . An alternative 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DHBD), designated EtbC, was identified in RDC1 cells grown on ethylbenzene . EtbC contained the broadest substrate specificity of any meta cleavage dioxygenase identified in a Rhodococcus strain to date, including RHA1 BphC . EtbC was purified to near homogeneity from RDC1 cells grown on ethylbenzene, and a 58-amino-acid NH2-terminal sequence was determined . The NH2-terminal amino acid sequence was used for the identification of the etbC gene from an RDC1 chromosomal DNA 2,3-DHBD expression library . The etbC gene was successfully cloned, and we report here the determination of its nucleotide sequence . The substrate specificity patterns of cell extract and native nondenaturing polyacrylamide gel electrophoresis analysis identified the coexpression of two 2,3-DHBDs (BphC and EtbC) in RHA1 cells grown on either biphenyl or ethylbenzene . The possible implication of coexpressed BphC extradiol dioxygenases in the strong polychlorinated-biphenyl degradation activity of RHA1 was suggested.

Mikrobiol Z, 1996 Jul-Aug, 58(4), 34 - 8
{The degradation of plasticizers by Rhodococcus erythropolis 40F}; Aleshchenkova ZM et al.; A Rhodococcus erythropolis 40 phi strain isolated from the soil utilizes 0.1-2% v/v dibutylphthalate (DBP) and di-(2-ethylhexyl)-phthalate (DEHP) as a single carbon source . The strain metabolizes DBP and DEHP via the following steps: diester --> phthalate --> benzoate --> p-hydroxybenzoate --> 3,4-dihydroxybvenzoate --> 3-ketoadipinate.

Eur J Clin Microbiol Infect Dis, 1996 Jul, 15(7), 588 - 94
Serologic responses to Rhodococcus equi in individuals with and without human immunodeficiency virus infection; Vullo V et al.; Thirty healthy blood donors, 15 workers from horse-breeding farms, 69 human immunodeficiency virus (HIV)-negative persons at risk for HIV infection, 125 HIV-infected subjects without Rhodococcus equi infection, and nine HIV-infected patients with Rhodococcus equi pneumonia were evaluated in order to detect serum antibodies to Rhodococcus equi precipitate-soluble antigen by an enzyme immunoassay (EIA) . Whereas EIA values for healthy donors, horse farm workers, individuals at risk for HIV infection, and HIV-positive subjects without Rhodococcus equi infection were comparable, HIV-infected patients with rhodococcal disease had significantly higher Rhodococcus equi antibody levels (p < 0.0001) . The clinical outcome of Rhodococcus equi pneumonia was more severe in subjects who had low levels of specific antibodies, whereas patients who recovered had elevated Rhodococcus equi antibody levels over time . Immunoblot studies showed that both Rhodococcus equi-infected patients and foals recognized a protein band of approximately 60 kDa in the Rhodococcus equi precipitate-soluble antigen . On the other hand, the Rhodococcus equi-infected patients did not react with the diffuse 15 to 17 kDa virulence-associated proteins that represent important virulence factors both in mice and horses.

J Vet Med Sci, 1996 Jul, 58(7), 669 - 72
Isolation of virulent and intermediately virulent Rhodococcus equi from soil and sand on parks and yards in Japan; Takai S et al.; Rhodococcus equi is an emerging opportunistic pathogen of human immunodeficiency virus-infected patients . However, little is known about the distribution of virulent and intermediately virulent R . equi in human environment . In the present study, R . equi was isolated from 173 of 234 (73.9%) samples collected from soil and sand on 115 parks and 49 yards in Japan . The numbers of R . equi from soil and sand ranged from 2.5 x 10(1) to 1.2 x 10(5) per gram of sample . None of 1,294 isolates from those samples showed virulence-associated 15- to 17-kDa antigens and a 20-kDa antigen . These results suggest that avirulent R . equi is widespread in parks and yards, but the human environment has not been contaminated with virulent and intermediately virulent R . equi strains yet.

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