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