J Microbiol Methods, 2002 Oct, 51(2), 181 - 9 Use of both 16S rRNA and engineered functional genes with real-time PCR to quantify an engineered, PCB-degrading Rhodococcus in soil; Rodrigues JL et al.; A real-time PCR (RTm-PCR) assay using fluorescently labeled oligonucleotides (TaqMan probes) was used to detect and quantify the recombinant Rhodococcus sp . strain RHA1(fcb) in soil . One primer and probe set targeted a hypervariable region of the 16S rRNA gene unique to strain RHA1(fcb) and its phylogenetic relatives, and the other set targeted the recombinant 4-chlorobenzoate (4-CBA) degradation operon (fcb) and was strain-specific . The method had a 6-log dynamic range of detection (10(2)-10(7) cells ml(-1)) for both probes when DNA from pure cultures was used . Although the method was less sensitive in soil, the estimated number of cells in soil by real-time PCR corresponded to the measured number of RHA1(fcb) cells determined by colony-forming units. Transpl Infect Dis, 2002 Mar, 4(1), 52 - 6 Rhodococcus equi infection in transplant recipients: a case of mistaken identity and review of the literature; Perez MG et al.; The incidence of Rhodococcus equi infection in solid-organ transplant recipients continues to rise throughout the world . Unfortunately, this opportunistic pathogen is still underestimated and potentially disregarded by physicians and microbiology laboratories due to its morphology on Gram staining . Pulmonary involvement is the most common finding in the immunocompromised host . We report a case of a 63-year-old heart-transplant recipient who presented with increasing fatigue and nonproductive cough for 3 weeks . After full evaluation, a lung abscess was demonstrated by thoracic computerized tomography (CT) . Blood and sputum cultures were remarkable for heavy "diphtheroids." Although the Gram-stain result was initially interpreted as a contaminant, a clinical suspicion for Rhodococcus assisted in further investigation . Broncheoalveolar lavage and CT-guided biopsy of the lung abscess revealed heavy growth of diphtheroids . However, further evaluation by a reference laboratory demonstrated mycolic acid staining consistent with R . equi . Surgical drainage and prolonged antibiotic therapy resulted in complete remission of the pneumonia and abscess . This represents the fourth reported case of R . equi infection in a heart transplant recipient . It is imperative that all physicians and laboratory staff consider R . equi when an immunocompromised patient has any type of pneumonia, especially with abscess formation. Transpl Infect Dis, 2002 Mar, 4(1), 46 - 51 Rhodococcus equi pulmonary infection in a pancreas-alone transplant recipient: consequence of intense immunosuppression; Lo A et al.; We report the case of a pancreas-alone transplant recipient who developed Rhodococcus equi pneumonia after receiving multiple courses of antilymphocyte therapy for the treatment of recurrent acute pancreas allograft rejection . We also review and discuss the diagnosis, clinical course, and treatment of 18 cases of R . equi infection reported in solid organ transplant recipients . The lung is the most common primary site of infection, but R . equi infection is difficult to diagnose because of the pleomorphic, gram-positive, and partially acid-fast nature of the organism . Treatment usually involves a combination of antibiotics including rifampin, macrolides, vancomycin, and ciprofloxacin . The optimal duration of therapy is unknown, but relapse is common if the duration of treatment is less than 14 days . The duration of therapy should be guided by clinical recovery, culture results, and radiographic findings . Monitoring levels of immunosuppressive agents-such as tacrolimus and cyclosporine-is needed in order to avoid clinically significant drug interactions with rifampin or the macrolides when these agents are used in order to treat R . equi infection in the transplant population. J Inorg Biochem, 2002 Jul 25, 91(1), 70 - 7 Site-directed mutagenesis for cysteine residues of cobalt-containing nitrile hydratase; Hashimoto Y et al.; Three cysteine residues, which are completely conserved among alpha-subunits in all nitrile hydratases, are thought to be the ligands of a metal ion in the catalytic center of this enzyme . These cysteine residues (i.e . alpha C102, alpha C105 and alpha C107) in the high-molecular-mass nitrile hydratase (H-NHase) of Rhodococcus rhodochrous J1 were replaced with alanine by site-directed mutagenesis using the R . rhodochrous ATCC12674 host-vector system, and the resultant transformants were investigated . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the cell-free extracts of each mutant transformant revealed that four mutant transformants (i.e . alpha C105A, alpha C107A, alpha C102A/C105A and alpha C105A/C107A) showed predominant alpha- and beta-subunit protein bands with a mobility identical to those of the native H-NHase, while three mutant transformants (i.e . alpha C102A, alpha C102A/C107A and alpha C102A/C105A/C107A) did not produce the corresponding proteins . The purified former four mutant enzymes showed neither enzymatic activity nor the maximum absorption at 410 nm which was detected in the wild type H-NHase . They also did not contain cobalt ions . Based upon these findings, these three cysteine residues were found to be essential for the active expression of H-NHase. Appl Microbiol Biotechnol, 2002 Jul, 59(2-3), 318 - 24 Epub 2002 May 09. Alkanotrophic Rhodococcus ruber as a biosurfactant producer; Philp JC et al.; In this report we examined the structure and properties of surface-active lipids of Rhodococcus ruber . Most historical interest has been in the glycolipids of Rhodococcus erythropolis, which have been extensively characterised . R . erythropolis has been of interest due to its great metabolic diversity . Only recently has the metabolic potential of R . ruber begun to be explored . One major difference in the two species is that most R . ruber strains are able to oxidise the gaseous alkanes propane and butane . In preparation for investigation of the effects of gas metabolism on biosurfactant production, we set out to characterise the biosurfactants produced during growth on liquid n-alkanes and to compare these with R . erythropolis glycolipids. Vet Pathol, 2002 Jan, 39(1), 132 - 6 Increased pulmonary activation of nuclear factor-kappaB (NF-kappaB) in foals inoculated with Rhodococcus equi is associated with increased expression of inflammatory cytokines; Uhl EW et al.; Previous studies revealed that foals inoculated with virulent Rhodococcus equi had significantly higher pulmonary levels of interleukin-1beta, interleukin-12 p40, interferon-gamma, and tumor necrosis factor-alpha mRNA compared to foals inoculated with an avirulent plasmid-cured derivative . The purpose of this study was to determine if the increases in cytokine expression were associated with increased pulmonary activation of nuclear factor-kappaB (NF-kappaB) . Electrophoretic mobility shift assays were performed on pulmonary nuclear protein extracted from foals treated with phosphate-buffered saline, or inoculated with either virulent or avirulent R . equi . NF-kappaB activation was increased in the nuclear extracts from foals inoculated with virulent R . equi at 14 days after inoculation when increased cytokine expression was also observed . Southwestern histochemistry revealed activated NF-kappaB in multinucleated giant cells that often contained bacteria . These results indicate that the cytokine response to R . equi is at least partially mediated by NF-kappaB activation. Appl Environ Microbiol, 2002 Jul, 68(7), 3279 - 86 Cloning and heterologous expression of an enantioselective amidase from Rhodococcus erythropolis strain MP50; Trott S et al.; The gene for an enantioselective amidase was cloned from Rhodococcus erythropolis MP50, which utilizes various aromatic nitriles via a nitrile hydratase/amidase system as nitrogen sources . The gene encoded a protein of 525 amino acids which corresponded to a protein with a molecular mass of 55.5 kDa . The deduced complete amino acid sequence showed homology to other enantioselective amidases from different bacterial genera . The nucleotide sequence approximately 2.5 kb upstream and downstream of the amidase gene was determined, but no indications for a structural coupling of the amidase gene with the genes for a nitrile hydratase were found . The amidase gene was carried by an approximately 40-kb circular plasmid in R . erythropolis MP50 . The amidase was heterologously expressed in Escherichia coli and shown to hydrolyze 2-phenylpropionamide, alpha-chlorophenylacetamide, and alpha-methoxyphenylacetamide with high enantioselectivity; mandeloamide and 2-methyl-3-phenylpropionamide were also converted, but only with reduced enantioselectivity . The recombinant E . coli strain which synthesized the amidase gene was shown to grow with organic amides as nitrogen sources . A comparison of the amidase activities observed with whole cells or cell extracts of the recombinant E . coli strain suggested that the transport of the amides into the cells becomes the rate-limiting step for amide hydrolysis in recombinant E . coli strains. Appl Environ Microbiol, 2002 Jul, 68(7), 3270 - 8 Monocyclic aromatic hydrocarbon degradation by Rhodococcus sp . strain DK17; Kim D et al.; Rhodococcus sp . strain DK17 was isolated from soil and analyzed for the ability to grow on o-xylene as the sole carbon and energy source . Although DK17 cannot grow on m- and p-xylene, it is capable of growth on benzene, phenol, toluene, ethylbenzene, isopropylbenzene, and other alkylbenzene isomers . One UV-generated mutant strain, DK176, simultaneously lost the ability to grow on o-xylene, ethylbenzene, isopropylbenzene, toluene, and benzene, although it could still grow on phenol . The mutant strain was also unable to oxidize indole to indigo following growth in the presence of o-xylene . This observation suggests the loss of an oxygenase that is involved in the initial oxidation of the (alkyl)benzenes tested . Another mutant strain, DK180, isolated for the inability to grow on o-xylene, retained the ability to grow on benzene but was unable to grow on alkylbenzenes due to loss of a meta-cleavage dioxygenase needed for metabolism of methyl-substituted catechols . Further experiments showed that DK180 as well as the wild-type strain DK17 have an ortho-cleavage pathway which is specifically induced by benzene but not by o-xylene . These results indicate that DK17 possesses two different ring-cleavage pathways for the degradation of aromatic compounds, although the initial oxidation reactions may be catalyzed by a common oxygenase . Gas chromatography-mass spectrometry and 300-MHz proton nuclear magnetic resonance spectrometry clearly show that DK180 accumulates 3,4-dimethylcatechol from o-xylene and both 3- and 4-methylcatechol from toluene . This means that there are two initial routes of oxidation of toluene by the strain . Pulsed-field gel electrophoresis analysis demonstrated the presence of two large megaplasmids in the wild-type strain DK17, one of which (pDK2) was lost in the mutant strain DK176 . Since several other independently derived mutant strains unable to grow on alkylbenzenes are also missing pDK2, the genes encoding the initial steps in alkylbenzene metabolism (but not phenol metabolism) appear to be present on this approximately 330-kb plasmid. J Bacteriol, 2002 Jul, 184(14), 3898 - 908 Identification of a new class of cytochrome P450 from a Rhodococcus sp; Roberts GA et al.; A degenerate set of PCR primers were used to clone a gene encoding a cytochrome P450 (the P450RhF gene) from Rhodococcus sp . strain NCIMB 9784 which is of unique primary structural organization . Surprisingly, analysis of the translation product revealed that the P450 is fused to a reductase domain at the C terminus which displays sequence conservation for dioxygenase reductase proteins . The reductase partner comprises flavin mononucleotide- and NADH-binding motifs and a {2Fe2S} ferredoxin-like center . The gene was engineered for heterologous expression in Escherichia coli, and conditions were found in which the enzyme was produced in a soluble form . A recombinant strain of E . coli was able to mediate the O dealkylation of 7-ethoxycoumarin in good yield, despite the absence of any recombinant redox proteins . This unprecedented finding leads us to propose that P450RhF represents the first example of a new class of cytochromes P450 in which the reducing equivalents are supplied by a novel reductase in a fused arrangement. J Ind Microbiol Biotechnol, 2002 Jul, 29(1), 20 - 7 Selection of microbial consortia for treating metal-working fluids; van der Gast CJ et al.; The aim of this research was to determine the effectiveness of a strategy for constructing microbial consortia for treating chemically mixed industrial effluent, based on a more thorough understanding of communities within waste metal-working fluids (MWFs) . Complementary phenotypic and genotypic methods revealed that the microbial communities in spent MWFs had low diversity and were very similar in species composition in samples originating from different locations and uses . Of 65 bacterial isolates studied, only 9 species were identified using fatty acid methyl ester (FAME) analysis . The results of genotypic analysis by denaturing gradient gel electrophoresis (DGGE) were congruent with observations made using FAME analysis . The metabolic potential of the isolates was assessed in terms of assimilation ability and tolerance of co-contaminants . The three isolates, selected (Clavibacter michiganensis, Methylobacterium mesophilicum, and Rhodococcus erythropolis) to form a consortium, were representative of three of the four most abundant populations and when combined could utilise or tolerate all of the individual MWF components, including the biocide and the recalcitrant compound benzotriazole. Appl Microbiol Biotechnol, 2002 Jun, 59(1), 62 - 7 Epub 2002 Apr 06. Cometabolic ring fission of dibenzofuran by Gram-negative and Gram-positive biphenyl-utilizing bacteria; Stope MB et al.; Thirty-five strains of soil bacteria were grown with biphenyl (BP) and tested for their capacity to cooxidize dibenzofuran (DBF) . During metabolism of DBF, the culture medium of 17 strains changed from colorless to orange, indicating a meta-cleavage pathway of DBF degradation . The ring cleavage product of these isolates was shown to be 2-hydroxy-4-(3'-oxo-3' H-benzofuran-2'-yliden)but-2-enoic acid (HOBB) . The strain SBUG 271, studied in detail and identified as Rhodococcus erythropolis, degraded DBF via 1,2-dihydroxydibenzofuran . The ensuing meta-cleavage yielded HOBB and salicylic acid . In addition, the four monohydroxylated monomers of DBF and two metabolites, which were not further characterized, were detected . Thus, our results demonstrate that the metabolic mechanism involves lateral dioxygenation of DBF followed by meta-cleavage and occurs in Gram-negative as well as in Gram-positive BP-degrading bacteria. J Biol Chem, 2002 Aug 30, 277(35), 31722 - 33 Epub 2002 Jun 18. A novel lipoarabinomannan from the equine pathogen Rhodococcus equi . Structure and effect on macrophage cytokine production; Garton NJ et al.; Rhodococcus equi is a major cause of foal morbidity and mortality . We have investigated the presence of lipoglycan in this organism as closely related bacteria, notably Mycobacterium tuberculosis, produce lipoarabinomannans (LAM) that may play multiple roles as virulence determinants . The lipoglycan was structurally characterized by gas chromatography-mass spectrometry following permethylation, capillary electrophoresis after chemical degradation, and (1)H and (31)P and two-dimensional heteronuclear nuclear magnetic resonance studies . Key structural features of the lipoglycan are a linear alpha-1,6-mannan with side chains containing one 2-linked alpha-d-Manp residue . This polysaccharidic backbone is linked to a phosphatidylinositol mannosyl anchor . In contrast to mycobacterial LAM, there are no extensive arabinan domains but single terminal alpha-d-Araf residue capping the 2-linked alpha-d-Manp . The lipoglycan binds concanavalin A and mannose-binding protein consistent with the presence of t-alpha-d-Manp residues . We studied the ability of the lipoglycans to induce cytokines from equine macrophages, in comparison to whole cells of R . equi . These data revealed patterns of cytokine mRNA induction that suggest that the lipoglycan is involved in much of the early macrophage cytokine response to R . equi infection . These studies identify a novel LAM variant that may contribute to the pathogenesis of disease caused by R . equi. J Biotechnol, 2002 Aug 7, 97(2), 163 - 76 Population analysis of a binary bacterial culture by multi-parametric flow cytometry; Muller S et al.; To study the degradation of a xenobiotic that requires a mixed culture it is essential to monitor the proportions and to control the population dynamics of the component strains . For these purposes fluorochromising techniques and multi-parametric flow cytometry were used to follow Rhodococcus erythropolis K2-3 and Ochrobactrum anthropi K2-14, both of which are needed to degrade 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB) . Although the two strains can grow in constant proportions in mixed cultures on other substrates, 2,4-DB could not be degraded as a sole substrate in a continuous process and R . erythropolis K2-3 was clearly impaired in the binary mixture . Addition of a second, easily assimilable substrate (xylitol) in appropriate concentrations (empirically determined) helped this strain survive, and thus facilitated complete degradation of the xenobiotic . This combination of substrates was found to stabilise the growth of R . erythropolis K2-3 and, consequently promoted the action of O . anthropi K2-14 . Thus, the two organisms became established in constant proportions in a continuous process until reaching steady state . Consequently, multiplication and cell division activities of the two components of the binary culture were high and reached similar values to those attained when they are grown in pure culture. Infect Immun, 2002 Jul, 70(7), 3768 - 76 H(2)O(2), which causes macrophage-related stress, triggers induction of expression of virulence-associated plasmid determinants in Rhodococcus equi; Benoit S et al.; The response of the intracellular pathogen Rhodococcus equi to H(2)O(2) treatment, a situation potentially encountered after the oxidative burst of alveolar macrophages, was analyzed . Compared to other bacteria, including Deinococcus radiodurans, R . equi showed exceptionally high resistance to this stress . A proteomic approach showed that four polypeptides present in the wild-type strain (85F) are missing in the plasmid-cured strain 85F(P-), and by using a DNA macroarray, we identified two plasmid-encoded vap genes, vapA and vapG, whose expression was highly induced by H(2)O(2) treatment . Whereas the transcript size of vapA was compatible with a monocistronic mRNA, the transcript of vapG was considerably longer . Rapid amplification of cDNA ends PCRs showed that the transcriptional start sites of the two operons were 69 and 269 nucleotides (nt) upstream of the start codon, respectively . Analysis of these leader sequences revealed the presence of a small open reading frame named podG, which encodes a sequence of 55 amino acids preceded by a putative ribosome binding site sequence in the vapG transcript . Taking this result into account, the untranslated leader of the podG/vapG operon is 87 nt . Alignment of this sequence with the leader sequences of vapA and vapD, genes previously shown to be induced by acid, revealed significant homologies . Since our results showed that vapA, vapD, and vapG are genes highly induced by macrophage-related stresses, their gene products may, within the Vap protein family, play a dominant role inside these phagocytic cells and may be the most promising candidates for vaccination strategies. Scand J Infect Dis, 2002, 34(4), 300 - 2 Rhodococcus equi brain abscess in an immunocompetent patient; Corne P et al.; Rhodococcus equi brain abscesses usually occur in immunocompromised patients with prolonged and refractory pulmonary infections . Herein we report a case of R . equi brain abscess in a 67-y-old man without immunodepression . Our patient recovered after neurosurgical resection and prolonged antimicrobial therapy with vancomycin and trimethoprim-sulfamethoxazole. J Bacteriol, 2002 Jul, 184(13), 3569 - 77 Substrate specificity of nickel/cobalt permeases: insights from mutants altered in transmembrane domains I and II; Degen O et al.; HoxN, a high-affinity, nickel-specific permease of Ralstonia eutropha H16, and NhlF, a nickel/cobalt permease of Rhodococcus rhodochrous J1, are structurally related members of the nickel/cobalt transporter (NiCoT) family . These transporters have an eight-helix structure and are characterized by highly conserved segments with polar or charged amino acid residues in transmembrane domains (TMDs) II, III, V, and VI . Two histidine residues in a Ni2+ binding motif, the signature sequence of NiCoTs, in TMD II of HoxN have been shown to be crucial for activity . Replacement of the corresponding His residues in NhlF affected both Co2+ and Ni2+ uptake, demonstrating that NhlF employs a HoxN-like mechanism for transport of the two cations . Multiple alignments of bacterial NiCoT sequences identified a striking correlation between a hydrophobic residue (Val or Phe) in TMD II and a position in the center of TMD I occupied by either an Asn (as in HoxN) or a His (as in NhlF) . Introducing an isoleucine residue at the latter position strongly reduced HoxN activity and abolished NhlF activity, suggesting that a Lewis base N-donor moiety is important . The Asn-to-His exchange had no effect on HoxN, whereas the converse replacement reduced NhlF-mediated Ni2+ uptake significantly . Replacement of the entire TMD I of HoxN by the respective NhlF segment resulted in a chimera that transported Ni2+ and Co2+ with low capacity . The Val-to-Phe exchange in TMD II of HoxN led to a considerable rise in Ni2+ uptake capacity and conferred to the variant the ability to transport Co2+ . NhlF activity dropped in response to the converse mutation . Our data predict that TMDs I and II in NiCoTs spatially interact to form a critical part of the selectivity filter . As seen for the V64F variant of HoxN, modification of this site can increase the velocity of transport and concomitantly reduce the specificity. Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 749 - 55 Transfer of Tsukamurella wratislaviensis Goodfellow et a . 1995 to the genus Rhodococcus as Rhodococcus wratislaviensis comb . nov.; Goodfellow M et al.; A polyphasic study was undertaken to clarify the taxonomic position of the type strain (N805T) of Tsukamurella wratislaviensis . This organism showed a combination of phenotypic properties, notably chemotaxonomic markers, consistent with its classification in the genus Rhodococcus . Comparative 16S rDNA sequencing studies indicated that strain 805T falls into the Rhodococcus erythropolis subclade, where it forms a monophyletic group with the type strains of Rhodococcus opacus and Rhodococcus percolatus . The close relationship between these strains was underpinned by the results of mycolic acid analyses . However, strain N805T was distinguished from the R . opacus and R . percolatus strains in DNA-DNA pairing experiments and by using a range of phenotypic properties . In light of these studies, it is clear that strain N805T is misclassified in the genus Tsukamurella . It is, therefore, proposed that Tsukamurella wratislaviensis Goodfellow et al . 1995 be transferred to the genus Rhodococcus as Rhodococcus wratislaviensis comb . nov.. J Biotechnol, 2002 Jul 17, 97(1), 1 - 11 Degradation of phenol by Rhodococcus erythropolis UPV-1 immobilized on Biolite in a packed-bed reactor; Begona Prieto M et al.; A strain of Rhodococcus erythropolis has been isolated and identified by 16S rRNA sequencing . Cells acclimated to phenol can be adsorbed on the external surface of beads of the ceramic support Biolite where they grow forming a network of large filaments . Exponentially-growing cells were adsorbed faster than their stationary-phase counterparts . Immobilization resulted in a remarkable enhancement of the respiratory activity of cells and a shorter lag phase preceding the active phenol degradation . Under optimum operation conditions, the immobilized cells in a laboratory-scale column reactor packed with support beads were able to degrade completely phenol in defined mineral medium at a maximum rate of 18 kg phenol m(-3) per day . The performance of the bioreactor in long-term continuous operation was characterized by pumping defined mineral medium which contained different concentrations of phenol at different flow-rates . Once phenol biodegradation in defined mineral medium was well established, an industrial wastewater from a resin manufacturing company, which contained both phenol and formaldehyde, was tested . In this case, after wastewater conditioning (i.e . pH, nitrogen source and micronutrient amendments) the immobilized cells were able to remove completely formaldehyde and to partly biodegrade phenols at a rate of 1 kg phenol m(-3) per day. Acta Crystallogr D Biol Crystallogr, 2002 Jun, 58(Pt 6 Pt 2), 1074 - 6 Epub 2002 May 29. 4-Chlorocatechol 1,2-dioxygenase from the chlorophenol-utilizing Gram-positive Rhodococcus opacus 1CP: crystallization and preliminary crystallographic analysis; Ferraroni M et al.; 4-Chlorocatechol 1,2-dioxygenase (4-ClC1,2DO) from the Gram-positive bacterium Rhodococcus opacus (erythropolis) 1CP, an enzyme involved in the aerobic biodegradation of chloroaromatic compounds, has been crystallized . 4-ClC1,2DO, which specifically catalyzes the intradiol cleavage of 4-substituted catechols, which are intermediates in the degradation of a variety of aromatic pollutants, to the corresponding maleylacetates, has recently been purified to homogeneity . The enzyme is an homodimer composed of two identical subunits in an alpha(2)-type quaternary structure; it has a molecular weight of about 29 kDa per monomer and contains one Fe(III) and one Mn(II) ion per homodimer . Hexagonal crystals grown in 1.6 M ammonium sulfate, 0.1 M sodium chloride, 100 mM Tris-HCl pH 9.0, 5-15% glycerol were successfully frozen under liquid nitrogen, adding 30% glycerol to the mother-liquor solution as a cryoprotectant . A complete data set was collected at 2.8 A resolution using the EMBL beamline BW7A at the DORIS storage ring, DESY, Hamburg, Germany with a MAR CCD detector and a wavelength of 1.01 A . The crystals belong to the primitive hexagonal space group P6(3)22, with unit-cell parameters a = 90.4, c = 307.5 A . This is the first intradiol dioxygenase which specifically catalyzes the cleavage of chlorocatechols in Gram-positive bacteria to give diffraction-quality crystals. Environ Microbiol, 2002 May, 4(5), 262 - 76 Discrimination and taxonomy of geographically diverse strains of nitrile-metabolizing actinomycetes using chemometric and molecular sequencing techniques; Brandao PF et al.; Mycolic acid-containing actinomycetes capable of metabolizing nitriles were recovered from deep-sea sediments and terrestrial soils by enrichment culture on acetonitrile, benzonitrile, succinonitrile or bromoxynil . A total of 43 nitrile-degrading strains were isolated and, together with previously recovered nitrile-degrading rhodococci, were identified by a polyphasic taxonomic approach, which included mycolic acid profiles, pyrolysis mass spectrometry (PyMS), genomic fingerprinting based on sequence variability of the 16S ribosomal RNA gene using polymerase chain reaction-restriction fragment length polymorphism-single-strand conformational polymorphism, and 16S rRNA gene sequence comparison . Isolates phylogenetically related to Rhodococcus erythropolis dominated the culturable microorganisms from most marine and terrestrial samples . These isolates clustered together in a major pyrogroup that showed high congruence with PRS profiles of the 16S rRNA gene . Such high congruence also was obtained for other recovered isolates that were assigned to species of Rhodococcus and Gordonia . Sequencing data validated the results obtained by PRS analysis and enabled phylogenetic relationships to be established . Some of the recovered bacteria probably represent novel microbial species . The fact that nitrile-metabolizing microorganisms were recovered from a wide range of habitat types suggests that nitrile transforming enzymatic activity is geographically widely distributed in nature. Can J Microbiol, 2002 Apr, 48(4), 295 - 304 Hydrophobicity development, alkane oxidation, and crude-oil emulsification in a Rhodococcus species; Bredholt H et al.; The relationship between the phenomena alkane oxidation, extreme hydrophobicity of the cell surface, and crude-oil emulsification in Rhodococcus sp . strain 094 was investigated . Compounds that induce the emulsifying ability simultaneously induced the cytochrome P450-containing alkane oxidizing system and the transition from low to high cell-surface hydrophobicity . Exposed to inducers of crude-oil emulsification, the cells developed a strong hydrophobic character during exponential growth, which was rapidly lost when entering stationary phase . The loss in hydrophobicity coincided in time with the crude-oil emulsification, indicating that the components responsible for the formation of cell-surface hydrophobicity act as excellent emulsion stabilisers only after release from the cells . Rhodococcus sp . strain 094 possessed three distinct levels of cell-surface hydrophobicity . One level of low hydrophobicity was characteristic of cells in late stationary phase and was independent of growth substrate . A second and more hydrophobic level was observed for cells in exponential phase grown on water-soluble substrates, while a third level, characterised by extreme cell hydrophobicity, was observed for cells in exponential phase cultivated on hydrophobic substrates such as hexadecane . The production of the oil-emulsifying agents seems to require external sources of nitrogen and phosphate. Mol Plant Microbe Interact, 2002 Apr, 15(4), 398 - 403 Virulence genes of the phytopathogen Rhodococcus fascians show specific spatial and temporal expression patterns during plant infection; Cornelis K et al.; The phytopathogenic bacterium Rhodococcus fascians provokes shoot meristem formation and malformations on aerial plant parts, mainly at the axils . The interaction is accompanied by bacterial colonization of the plant surface and tissues . Upon infection, the two bacterial loci required for full virulence, fas and att, were expressed only at the sites of symptom development, although their expression profiles differed both spatially and temporally . The att locus was expressed principally in bacteria located on the plant surface at early stages of infection . Expression of the fas locus occurred throughout infection, mainly in bacteria that were penetrating, or had penetrated, the plant tissues and coincided with sites of meristem initiation and proliferation . The implications for the regulation of virulence genes of R . fascians during plant infection are discussed. Mikrobiologiia, 2002 Mar-Apr, 71(2), 166 - 70 {The role of low-molecular-weight nitrogen compounds in the osmotolerance of Rhodococcus erythropolis and Arthrobacter globiformis}; Komarova TI et al.; Investigations showed that Rhodococcus erythropolis E-15 and Arthrobacter globiformis 2F cells respond to osmotic shock by increasing the synthesis of free amino acids, primarily glutamic acid (80% of the intracellular free amino acid pool) . The osmoprotective role of glutamic acid follows from its beneficial effect on the growth of bacteria in high-salinity media . It was found that the addition of this amino acid to the growth medium at a concentration of 2 mM shortened the lag phase and increased the growth rate and biomass yield of either of the two bacteria . The addition of another osmoprotectant, trehalose, to the high-salinity growth medium of R . erythropolis E-15 at the same concentration (2 mM), restored the growth parameters of this bacterium to the control values. J Appl Microbiol, 2002, 92(6), 1058 - 65 Survival and naphthalene-degrading activity of Rhodococcus sp . strain 1BN in soil microcosms; Cavalca L et al.; AIMS: The survival and activity of Rhodococcus sp . strain 1BN, inoculated into naphthalene-contaminated sandy-loam soil microcosms, were studied using classical and molecular methods . METHODS AND RESULTS: The naphthalene-degrading activity of 1BN in microcosms was examined through viable counts, CO2 production and naphthalene consumption, while its survival after inoculation was monitored by detecting the contemporary presence of alkane and naphthalene degradative genes and by analysing the 16S rDNA specific restriction profile . The inoculation of 1BN did not significantly enhance naphthalene degradation in the naphthalene-contaminated native soil, where 1BN maintained its catabolic activity also when in the presence of indigenous microflora . Instead the rate of naphthalene degradation by the inoculated 1BN was greater in sterile naphthalene-contaminated soil . The level of 1BN was only slightly higher after inoculation regardless of whether indigenous naphthalene-degrading bacteria were present or not and 1BN remained viable even when the substrate was depleted . CONCLUSIONS: This study documents the colonization and growth of 1BN in a non-sterile, naphthalene-added, sandy-loam soil having an active indigenous naphthalene-degrading population . SIGNIFICANCE AND IMPACT OF THE STUDY: An active and well-established naphthalene-degrading bacterial population in the native soil did not hamper the survival of the introduced 1BN that, through its activity, enhanced the mineralization rate of naphthalene. FEMS Microbiol Lett, 2002 Apr 9, 209(2), 307 - 12 Reductive deamination as a new step in the anaerobic microbial degradation of halogenated anilines; Travkin V et al.; In this paper we report the isolation and characterization of an anaerobic enrichment culture as well as of a Rhodococcus sp . strain 2 capable of degrading 3,4-dihaloanilines under nitrate reducing conditions . Using mass spectrometry several of the intermediates formed in the process of 3,4-dichloroaniline conversion were identified . Most interesting is the observation of reductive deamination and the formation of 1,2-dichlorobenzene as one of the intermediates . Using 19F NMR and fluorinated 3,4-dihaloaniline model substrates it was corroborated that reductive deamination of the anilines to give dihalobenzene intermediates represents a new initial step in the anaerobic microbial degradation of these halogenated anilines. J Vet Pharmacol Ther, 2002 Apr, 25(2), 99 - 104 Pharmacokinetics of azithromycin in foals after i.v . and oral dose and disposition into phagocytes; Davis JL et al.; The properties of azithromycin suggest that it may be an alternative to erythromycin for treatment of Rhodococcus equi pneumonia in foals . To investigate this possibility, the disposition of azithromycin in plasma, polymorphonuclear leukocytes (PMN), and alveolar cells was examined after a single administration in foals . Azithromycin suspension was administered orally (p.o.) at a dose of 10 mg/kg to five healthy 2-3-month-old foals . Two weeks later, azithromycin for injection was administered by intravenous (i.v.) infusion at a dose of 5 mg/kg to the same foals . Plasma samples were collected after p.o . and i.v . administration . Peripheral blood PMN and bronchoalveolar lavage fluid and alveolar cells were collected after p.o . administration . Azithromycin concentrations were determined by reverse-phase high-performance liquid chromatography (HPLC) with coulometric electrochemical detection . Azithromycin p.o . absorption was variable with a mean systemic availability of 39% (+/-20%) . The plasma half-life was 16 and 18.3 h after i.v . and p.o . administration, respectively . Azithromycin had a very large volume of distribution (V(d)) of 11.6 L/kg {V(d(ss))} and 12.4 L/kg {V(d(area))} . The large V(d) can be attributed to high tissue and intracellular concentrations, exhibited by the high concentration of azithromycin in PMN and alveolar cells . The PMN half-life was 49.2 h . Dosage of 10 mg/kg of azithromycin p.o . once daily for foals with R . equi pneumonia is recommended for further study. Biodegradation, 2001, 12(5), 317 - 23 Isobutylidenediurea degradation by Rhodococcus erythropolis; Jahns T et al.; A new enzyme (isobutylidenediurea amidinohydrolase) catalyzing the hydrolysis of isobutylidenediurea (a condensation product of urea and isobutyraldehyde widely used as a slow-release nitrogeneous fertilizer) was characterized from a strain of Rhodococcus erythropolis . The enzyme was purified 1,250-fold to apparent homogeneity and shown to hydrolyze the fertilizer to urea and isobutyraldehyde at a molar ratio of 2: 1 . No activity was observed with ureido- or other structurally related compounds . Its molecular mass was determined by native polyacrylamide gelelectrophoresis and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry to be 15 kDa (+/-2 kDa) and 16.4 kDa, respectively . Growth of the bacterium in the presence of isobutylidenediurea led to an increased expression of the constitutively synthetized enzyme. Microbiology, 2002 May, 148(Pt 5), 1581 - 91 Formation and resuscitation of "non-culturable" cells of Rhodococcus rhodochrous and Mycobacterium tuberculosis in prolonged stationary phase; Shleeva MO et al.; After growth of Rhodococcus rhodochrous in Sauton's medium, and further incubation for about 60 h in stationary phase, there was a transient (up to 5 log) decrease in the c.f.u . count, whereas the total count remained similar to its initial value . At the point of minimal viability, the most probable number (MPN) count was 10 times greater than the c.f.u . count . This difference was further magnified by 3-4 logs (giving values close to the total count) by incorporating supernatant taken from growing cultures . A small protein similar to Rpf (resuscitation-promoting factor of Micrococcus luteus) appeared to be responsible for some of the activity in the culture supernatant . The formation of "non-culturable" cells of the "Academia" strain of Mycobacterium tuberculosis was similarly observed following growth in Sauton's medium containing Tween 80 in sealed culture vessels, and further incubation for an extended stationary phase . This resulted in the formation, 4-5 months post-inoculation, of a homogeneous population of ostensibly "non-culturable" cells (zero c.f.u.) . Remarkably, the MPN count for these cultures was 10(5) organisms ml(-1), and this value was further increased by one log using supernatant from an actively growing culture . Populations of "non-culturable" cells of Mycobacterium tuberculosis were also obtained by the filtration of "clumpy" cultures, which were grown in the absence of Tween 80 . These small cells could only be grown in liquid medium (MPN) and their viability was enhanced by the addition of culture supernatant or Rpf . The "non-culturable" cells that accumulated during prolonged stationary phase in both the R . rhodochrous and the Mycobacterium tuberculosis cultures were small ovoid and coccoid forms with an intact permeability barrier, but with undetectable respiratory activity . The authors consider these non-culturable bacteria to be dormant . The observed activity of culture supernatants and Rpf with "non-culturable" bacterial suspensions invites the speculation that one, or more, of the cognate Mycobacterium tuberculosis Rpf-like molecule(s) could be involved in mechanisms of latency and reactivation of tuberculosis in vivo. Microbiology, 2002 May, 148(Pt 5), 1407 - 12 Identification of phenyldecanoic acid as a constituent of triacylglycerols and wax ester produced by Rhodococcus opacus PD630; Alvarez HM et al.; Phenyldecane supported growth and lipid accumulation of Rhodococcus opacus PD630 during cultivation under nitrogen-limiting conditions . The results of this study suggested that the hydrocarbon phenyldecane was degraded by monoterminal oxidation, followed by beta-oxidation of the alkyl side-chain to phenylacetic acid, and by an additional degradative route for the oxidation of the latter to intermediates of the central metabolism . alpha-Oxidation of phenyldecanoic acid also occurred to some extent . Phenyldecanoic acid, the monoterminal oxidation product, was also utilized for the biosynthesis of a novel wax ester and novel triacylglycerols . The formation of the wax ester phenyldecylphenyldecanoate probably resulted from the condensation of phenyldecanoic acid and phenyldecanol, which were produced as metabolites during the catabolism of phenyldecane . Two types of triacylglycerol were detected in phenyldecane-grown cells of strain PD630 . Triacylglycerols containing only odd- and even-numbered aliphatic fatty acids, as well as triacylglycerols in which one fatty acid was replaced by a phenyldecanoic acid residue, occurred . Other phenyl intermediates, such as phenylacetic acid, phenylpropionic acid, 4-hydroxyphenylpropionic acid, protocatechuate and homogentisic acid, were excreted into the medium during cultivation on phenyldecane . On the basis of the results obtained, pathways for the catabolism and assimilation of phenyldecane by R . opacus PD630 are discussed. Clin Infect Dis, 2002 May 15, 34(10), 1379 - 85 Epub 2002 Apr 25. Rhodococcus equi: an emerging pathogen; Weinstock DM et al.; More than 100 cases of Rhodococcus equi infection have been reported since the first description of human disease caused by this organism . The vast majority of patients infected with R . equi are immunocompromised, and two-thirds have human immunodeficiency virus infection . The clinical manifestations of R . equi infection are diverse, although 80% of patients have some pulmonary involvement . The organism is easily cultured from specimens of infected tissue or body fluid, but it may be misdiagnosed as a contaminant . Treatment is often prolonged, and relapses at distant sites are common . This article summarizes the history, diagnosis, clinical features, and treatment of infection with this emerging pathogen. Appl Environ Microbiol, 2002 May, 68(5), 2337 - 43 Extracellular polysaccharides of Rhodococcus rhodochrous S-2 stimulate the degradation of aromatic components in crude oil by indigenous marine bacteria; Iwabuchi N et al.; Rhodococcus rhodochrous S-2 produces extracellular polysaccharides (S-2 EPS) containing D-glucose, D-galactose, D-mannose, D-glucuronic acid, and lipids, which is important to the tolerance of this strain to an aromatic fraction of (AF) Arabian light crude oil (N . Iwabuchi, N . Sunairi, H . Anzai, M . Nakajima, and S . Harayama, Appl . Environ . Microbiol . 66:5073-5077, 2000) . In the present study, we examined the effects of S-2 EPS on the growth of indigenous marine bacteria on AF . Indigenous bacteria did not grow significantly in seawater containing AF even when nitrogen, phosphorus, and iron nutrients were supplemented . The addition of S-2 EPS to seawater containing nutrients and AF resulted in the emulsification of AF, promotion of the growth of indigenous bacteria, and enhancement of the degradation of AF by the bacteria . PCR-denaturing gradient gel electrophoresis analyses show that addition of S-2 EPS to the seawater containing nutrients and AF changed the composition of the bacterial populations in the seawater and that bacteria closely related to the genus Cycloclasticus became the major population . These results suggest that Cycloclasticus was responsible for the degradation of hydrocarbons in AF . The effects of 15 synthetic surfactants on the degradation of AF by indigenous marine bacteria were also examined, but enhancement of the degradation of AF was not significant . S-2 EPS was hence the most effective of the surfactants tested in promoting the biodegradation of AF and may thus be an attractive agent to use in the bioremediation of oil-contaminated marine environments. Prikl Biokhim Mikrobiol, 2002 Mar-Apr, 38(2), 136 - 9 {Bacterial succession on n-alkanes under the conditions of sulfate reduction}; Koronelli TV et al.; The dynamics of species composition of a hydrocarbon-oxidizing bacteriocenosis of a ground suspension of Mozhaisk Reservoir has been studied . The bacteriocenosis was undergoing development in a paraffin film (model association composed of sulfate-reducing bacteria and hydrocarbon-oxidizing bacteria) . The type of bacterial succession did not depend on the depth, from which ground samples were collected . Two microbial species (Pseudomonas sp . and Arthrobacter globiformis) were absolutely dominant . Pseudomonas sp . was dominant at the early and intermediate stages of the succession, whereas A . globiformis was present in the hydrocarbon-oxidizing bacteriocenosis throughout the whole period of the succession . There was a trend toward a gradual increase in the ratio of A . globiformis, and, by the end of the experiment, Pseudomonas sp . was replaced by A . globiformis almost completely . The bacterial species Micrococcus sp . and Rhodococcus erythropolis were minor components of the hydrocarbon-oxidizing bacteriocenosis under the conditions of sulfate reduction . The succession of species of hydrocarbon-oxidizing bacteria in the paraffin film of the model association reflects both the life strategy of the bacterial species under study and the degree of their tolerance to products of sulfate reduction. J Vet Diagn Invest, 2002 Mar, 14(2), 157 - 9 Placentitis, fetal pneumonia, and abortion due to Rhodococcus equi infection in a Thoroughbred; Patterson-Kane JC et al.; Rhodococcus equi is a rare cause of equine abortion . This report describes pyogranulomatous placentitis and fetal pneumonia in a case of abortion from a Thoroughbred mare . Numerous Gram-positive coccobacilli were noted histologically within macrophages in placental and pulmonary lesions . Rhodococcus equi was isolated in pure culture from the placenta, lung, liver, kidney, and stomach content . This is the first description of placentitis due to Rhodococcus equi infection in a horse. Ann Pathol, 2002 Feb, 22(1), 56 - 9 {Role of cytology in the diagnosis of Rhodococcus equi infection}; Hofman V et al.; A 59-year-old HIV-positive man developed necrotizing right-lobe pneumonia with abscess formation . Bronchial aspirates and bronchoalveolar lavage showed numerous inflammatory cells, mainly histiocytes and neutrophils, and intracellular and extracellular Gram- and Ziehl-Nielsen-positive cocci and coccobacilli . Rhodococcus equi was identified . The patient died one week after diagnosis and necropsy confirmed the presence of R . equi nectrotizing pneumonia. Int J Syst Evol Microbiol, 2002 Mar, 52(Pt 2), 409 - 13 Rhodococcus jostii sp . nov., isolated from a medieval grave; Takeuchi M et al.; The taxonomic position of a bacterial strain isolated from the femur of the remains of Jost Lucembursky, margrave in Moravia, Brno (Czech Republic), was investigated by phenotypic, chemotaxonomic and molecular taxonomic methods . The chemotaxonomic characteristics, including the cell-wall amino acid and sugar compositions, the quinone system and the fatty acid profile, were in good agreement with those of the genus Rhodococcus . The G+C content of the DNA was 67.4 mol% . Comparative 16S rRNA gene sequencing demonstrated that the unknown strain represents a distinct line of descent within the genus Rhodococcus . The nearest relatives of the bacterium were Rhodococcus opacus and Rhodococcus percolatus . The unknown bacterium was readily distinguished from these species by using phenotypic methods . On the basis of phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as Rhodococcus jostii sp . nov . The type strain is strain IFO 16295T (= CCM 4760T). Biotechnol Appl Biochem, 2002 Apr, 35(Pt 2), 69 - 74 Growth and production of cholesterol oxidase by alginate-immobilized cells of Rhodococcus equi No . 23; Chang YC et al.; Rhodococcus equi No . 23 was immobilized in calcium alginate . No detrimental effect on the viability of the test organism was observed during the immobilization procedure . Approx . 98% of the cell population originally present in the alginate solution were immobilized in the gel beads . When the cells of an equal volume of the culture, obtained respectively at exponential phase (12 h preculture), late-exponential phase (20 h preculture) or stationary phase (36 h preculture) were immobilized, the gel beads prepared with the stationary-phase culture were found to contain the highest cell population {about 10(8) colony-forming units (CFU)/g of beads} . In addition, gel beads, prepared with late-exponential-phase culture, exhibited the highest production of cholesterol oxidase (CholOx) after 48 h of incubation . Increasing the bead mass from 3.5 to 14.0 g/100 ml of medium increased CholOx production . However, further increasing the bead mass resulted in a reduction of CholOx production . Furthermore, on the basis of a similar initial cell population, the alginate-immobilized cells of R . equi No . 23 produced a significantly higher amount of CholOx (P<0.05) than did the free cells. J Small Anim Pract, 2002 Mar, 43(3), 129 - 32 Pyogranulomatous skin disease and cellulitis in a cat caused by Rhodococcus equi; Patel A; This report describes a case of Rhodococcus equi infection causing pyogranulomatous skin disease and cellulitis in a two-year-old female domestic shorthaired cat . The case differed from previously reported cases in cats in its clinical presentation and in the locations of the lesions, which were similar to those seen in horses . The presence of an intracellular organism was confirmed by cytology and on histopathology . The aetiological diagnosis was confirmed by routine biochemical tests specific for R . equi on a pure isolate obtained from a biopsy specimen . The report also reviews the literature of the documented feline cases and discusses the common pitfalls in the diagnosis of such infections. BMC Biotechnol . 2002 Mar 26;2(1):3. Rhodococcus erythropolis ATCC 25544 as a suitable source of cholesterol oxidase: cell-linked and extracellular enzyme synthesis, purification and concentration; Sojo MM et al.; BACKGROUND: The suitability of the strain Rhodococcus erythropolis ATCC 25544 grown in a two-liter fermentor as a source of cholesterol oxidase has been investigated . The strain produces both cell-linked and extracellular cholesterol oxidase in a high amount, that can be extracted, purified and concentrated by using the detergent Triton X-114 . RESULTS: A spray-dry method of preparation of the enzyme inducer cholesterol in Tween 20 was found to be superior in both convenience and enzyme synthesis yield to one of heat-mixing . Both were similar as far as biomass yield is concerned . Cell-linked cholesterol oxidase was extracted with Triton X-114, and this detergent was also used for purification and concentration, following temperature-induced detergent phase separation . Triton X-114 was utilized to purify and to concentrate the cell-linked and the extracellular enzyme . Cholesterol oxidase was found mainly in the resulting detergent-rich phase . When Triton X-114 concentration was set to 6% w/v the extracellular, but not the cell-extracted enzyme, underwent a 3.4-fold activation after the phase separation process . This result is interpreted in the light of interconvertible forms of the enzyme that do not seem to be in equilibrium . Fermentation yielded 360 U/ml (672 U/ml after activation), 36% of which was extracellular (65% after activation) . The Triton X-114 phase separation step yielded 11.6-fold purification and 20.3-fold concentration . CONCLUSIONS: The results of this work may make attractive and cost-effective the implementation of this bacterial strain and this detergent in a purification-based industrial production scheme of commercial cholesterol oxidase. Arch Microbiol, 2002 Mar, 177(3), 274 - 8 Epub 2002 Jan 08. A novel Streptomyces gene, samR, with different effects on differentiation of Streptomyces ansochromogenes and Streptomyces coelicolor; Tan H et al.; A 1.4-kb DNA fragment from Streptomyces ansochromogenes accelerated mycelium formation of S . ansochromogenes when present on a multicopy plasmid . The DNA fragment contains one complete open reading frame, designated samR, encoding a protein with 213 amino acids that contains a likely DNA-binding helix-turn-helix motif close to its N-terminus . The deduced SamR protein resembles the product of the hppR gene, which is involved in the regulation of catabolism of 3-(3-hydroxyphenyl) propionate in Rhodococcus globerulus . A samR disruption mutant was constructed that presented a bald phenotype and failed to form aerial hyphae and spores . We suggest that samR plays an important role in the emergence of aerial hyphae from substrate mycelium . An almost identical gene of Streptomyces coelicolor was also subjected to gene disruption . Surprisingly, the mutant was able to develop an aerial mycelium, but it remained white and deficient in sporulation instead of forming gray spores. Microbiology, 2002 Mar, 148(Pt 3), 799 - 806 npd gene functions of Rhodococcus (opacus) erythropolis HL PM-1 in the initial steps of 2,4,6-trinitrophenol degradation; Heiss G et al.; Rhodococcus (opacus) erythropolis HL PM-1 grows on 2,4,6-trinitrophenol (picric acid) or 2,4-dinitrophenol (2,4-DNP) as sole nitrogen source . A gene cluster involved in picric acid degradation was recently identified . The functional assignment of three of its genes, npdC, npdG and npdI, and the tentative functional assignment of a fourth one, npdH, is reported . The genes were expressed in Escherichia coli as His-tag fusion proteins that were purified by Ni-affinity chromatography . The enzyme activity of each protein was determined by spectrophotometry and HPLC analyses . NpdI, a hydride transferase, catalyses a hydride transfer from reduced F420 to the aromatic ring of picric acid, generating the hydride sigma-complex (hydride Meisenheimer complex) of picric acid (H(-)-PA) . Similarly, NpdI also transformed 2,4-DNP to the hydride sigma-complex of 2,4-DNP . A second hydride transferase, NpdC catalysed a subsequent hydride transfer to H(-)-PA, to produce a dihydride sigma-complex of picric acid (2H(-)-PA) . All three reactions required the activity of NpdG, an NADPH-dependent F420 reductase, for shuttling the hydride ions from NADPH to F420 . NpdH converted 2H(-)-PA to a hitherto unknown product, X . The results show that npdC, npdG and npdI play a key role in the initial steps of picric acid degradation, and that npdH may prove to be important in the later stages. Microbiology, 2002 Mar, 148(Pt 3), 793 - 8 Isocitrate lyase of the facultative intracellular pathogen Rhodococcus equi; Kelly BG et al.; Isocitrate lyase is the first enzyme of the glyoxylate shunt which is required for the assimilation of fatty acids and acetate . The intracellular pathogen Rhodococcus equi contains high activities of this enzyme following growth on acetate and lactate, indicating that it plays an important role in the metabolism of these substrates . The gene encoding isocitrate lyase (aceA) was cloned and sequenced . It specifies a 46846 Da protein, which was shown to be functional by expressing it in Escherichia coli . A gene similar to fadB, encoding 3-hydroxyacyl-CoA dehydrogenase, was located 90 bp downstream from aceA . Northern hybridization and RT-PCR experiments showed that aceA and fadB are cotranscribed into a 2.8 kb transcript . A smaller 1.6 kb aceA transcript was also observed which was 2.5-fold more abundant than the aceA-fadB transcript . It is proposed that a stable hairpin structure with a free energy (DeltaG) of -28.5 kcal x mol(-1) and located in the 90 bp aceA-fadB intergenic region is involved in stabilizing the aceA transcript. Appl Microbiol Biotechnol, 2002 Feb, 58(2), 260 - 3 Formaldehyde removal in synthetic and industrial wastewater by Rhodococcus erythropolis UPV-1; Hidalgo A et al.; Rhodococcus erythropolis strain UPV-1 is able to grow on phenol as the only carbon and energy source and to remove formaldehyde completely from both synthetic and industrial wastewater . The rate of formaldehyde removal is independent of either initial biomass or formaldehyde concentration . The presence of viable, intact cells is strictly necessary for this removal to take place . Discontinuous and continuous formaldehyde-feed systems were successfully tested with synthetic wastewater in shaken flasks . Once biodegradation was well established in model synthetic wastewater, a real wastewater sample was obtained from a local phenolic and melamine resin-manufacturing company . Incubation of biomass with this wastewater at subtoxic concentrations of formaldehyde resulted in the complete removal of the pollutant . Parameters, such as chemical oxygen demand and toxicity, were assessed as indicators of wastewater cleanup progress. Clin Microbiol Infect, 1996 Jun, 1(4), 230 - 234 Aseptic meningitis after neurosurgery: a demonstration of bacterial involvement; Druel B et al.; OBJECTIVE: To evaluate the presence of bacteria in samples from patients suffering from 'aseptic' meningitis following craniotomy . METHODS: Prospective study in which cerebrospinal fluid (CSF) from patients suffering from post-craniotomy meningitis and negative control patients were submitted to conventional culture and to polymerase chain reaction (PCR) using bacterial 16S rRNA universal primers, followed in some cases by DNA sequencing of the PCR product and phylogenetic analysis . RESULTS: CSF from patients with either culture-positive or culture-negative meningitis yielded positive amplifications, whereas no amplification was obtained with CSF from control patients . All positive signals were confirmed by Southern hybridization with a prokaryote 16S RNA-specific probe . Six PCR products, of which three were collected from later cases of culture-negative meningitis, were cloned and sequenced . Sequence analysis suggested affinities with Pseudomonas in three cases, with Escherichia in two cases and with Rhodococcus in one case . CONCLUSIONS: Many cases of culture-negative (aseptic) meningitis are probably bacterial meningitis and justify antibiotic treatment . The bacteria responsible for these cases of culture-negative meningitis might have peculiar growth requirements in vitro. Clin Microbiol Infect, 1995 Sep, 1(1), 18 - 23 Rhodococcus equi Virulence-Associated Antigens and Specific Antibody Response in AIDS Patients Infected with R . equi; Mastroianni CM et al.; OBJECTIVES: To analyze the expression of the 15- to 17-kDa plasmid-encoded antigens from Rhodococcus equi isolates of 7 AIDS patients and determine the immunologic response to these proteins in the patients' sera . METHODS: The expression of the virulence proteins in R . equi isolates and the specific antibody response were investigated by immunoblotting . Plasmid DNA was analyzed by agarose gel electrophoresis . RESULTS: The only patient infected with a strain carrying the virulence 85-kb plasmid and expressing the 15- to 17-kDa antigens developed a fatal pneumonia and did not produce specific antibodies to the virulence proteins . Of the 6 patients infected with R . equi strains lacking both proteins and plasmid, only 1 subject had a pulmonary disease with poor clinical outcome and exhibited a negligible humoral immune response to R . equi antigenic components, whereas the other patients who produced a remarkable antibody response developed either an asymptomatic infection (1 case) or pneumonia (4 cases) which completely cleared up . CONCLUSIONS: Our findings suggest that R . equi disease can be induced without the expression of the 15- to 17-kDa virulence-associated plasmid-encoded antigens in HIV-infected patients with very low CD4+ cell counts . Nevertheless, both the synthesis of the virulence proteins and a defective humoral immune response to R . equi may contribute to the severity of rhodococcal disease. Clin Microbiol Infect, 1997 Feb, 3(1), 12 - 18 Rhodococcus equi infection in non-HIV-infected patients . Two case reports and review; Farina C et al.; OBJECTIVE: To review two recent cases in HIV-negative subjects in the light of literature reports (52 patients without HIV infection till 1994) . METHODS: Epidemiology (animal contacts, risk factors, year, country), clinical presentation, diagnostic methods (X-ray, tomography, microbiological techniques), therapeutic approach (antibiotics, surgery) and outcome were evaluated on the basis of clinical literature reports . RESULTS: Tumors constituted an important predisposing factor and less frequently hepatobiliary pathology, rheumatologic diseases, iatrogenic causes, psychiatric pathology and trauma . Exposure to animals was reported by 55% of the patients . Pneumonia and pleurisy, without preferential localization, were detected in 50% of the patients . Etiologic diagnosis was usually obtained after an invasive collection . Combined medical therapy and surgery were required by 27.8% of the patients, and 16.7% of the patients died . CONCLUSIONS: In recent years the number of Rhodococcus equi cases has been rising also in HIV-negative patients . The infection is ubiquitous . Accurate diagnosis and the prompt selection of the most appropriate therapy depend on close cooperation between clinicians and microbiologists. Proc Natl Acad Sci U S A, 2002 Feb 19, 99(4), 1801 - 6 Optimizing bioconversion pathways through systems analysis and metabolic engineering; Stafford DE et al.; We demonstrate a general approach for metabolic engineering of biocatalytic systems comprising the uses of a chemostat for strain improvement and radioisotopic tracers for the quantification of pathway fluxes . Flux determination allows the identification of target pathways for modification as validated by subsequent overexpression of the corresponding gene . We demonstrate this method in the indene bioconversion network of Rhodococcus modified for the overproduction of 1,2-indandiol, a key precursor for the AIDS drug Crixivan. J Vet Med B Infect Dis Vet Public Health, 2001 Dec, 48(10), 751 - 8 Rapid immunohistochemical detection of Rhodococcus equi in impression smears from affected foals on postmortem examination; Szeredi L et al.; The first objective of this study was to develop an immunohistochemical procedure for rapid detection of Rhodococcus equi in impression smears from affected organs of foals on postmortem examination . The second aim was to demonstrate whether R . equi can be detected in smears of tracheal exudates collected from the same foals using an immunohistochemical method . Impression smears and cryostat and paraffin-embedded sections were made from the lungs and mediastinal lymph nodes of three foals (A, B and C) that had died of respiratory disease caused by R . equi, and also from the caudal mesenteric lymph node of foal A . Impression smears were made from the tracheal exudates of all foals . An affinity purified rabbit IgG was used for the immunohistochemical demonstration of R . equi . This antibody reacted with serotype 1 of R . equi in Ouchterlony's immunodiffusion and in the passive haemagglutination test, but not with other serotypes or with Streptococcus equi ssp . equi or Staphylococcus aureus, and failed to give an immunohistochemical reaction with Mycobacterium bovis or M . paratuberculosis . The immunohistochemical method proved to be of identical sensitivity to bacterial culture; moreover, from the lungs and mediastinal lymph nodes of one foal, R . equi could only be detected by this method . R . equi was demonstrated in smears of the tracheal exudates of all three foals . The results of this study indicate that the immunohistochemical method may be used for the rapid detection of R . equi in impression smears from the affected organs, especially abscesses, obtained postmortem, and possibly as a tool for diagnosing R . equi pneumonia in live foals by examining smears of tracheal aspirates. Am J Kidney Dis . 2002 Feb;39(2):E7. Medical management of pneumonia caused by Rhodococcus equi in a renal transplant recipient; Gonzalez-Roncero FM et al.; Rhodococcus equi is an animal pathogen that occasionally causes opportunistic infections in immunocompromised patients . The most common clinical picture is one of necrotizing pneumonia with a tendency toward cavitation and the formation of abscesses . We report a case of pneumonia caused by R equi in a renal transplant patient . An excellent response was shown to antibiotic treatment . Symptoms regressed, and the progressive disappearance of the lesion was confirmed on follow-up computed tomography scans . Surgical intervention or other invasive procedures were not required . To our knowledge, 14 cases of infection by R equi in solid-organ transplant patients have been described to date . Nine were recipients of a renal allograft . Surgery was required in many of these patients, and all the renal transplant recipients required the use of invasive therapeutic techniques, such as pleural drainage . This is the first case of a renal transplant recipient in whom radiologic presentation was as a solid nodule without ensuing cavitation that resolved exclusively with antibiotic treatment. Biotechnol Appl Biochem, 2002 Feb, 35(Pt 1), 61 - 7 Enhancement of enzyme activity and enantioselectivity via cultivation in nitrile metabolism by Rhodococcus sp . CGMCC 0497; Wu ZL et al.; Racemic 2-phenylpropionitrile was resolved enantioselectively by nitrile-converting enzymes in cells of Rhodococcus sp . CGMCC 0497 to S-(+)-2-phenylpropionic acid and R-(-)-2-phenylpropionamide . By optimization of the culture conditions, great enhancement of enzyme activity and enantioselectivity was achieved . Furthermore, the relationship between cell-growth periodicity and enzyme accumulation was studied; the addition of inducer was delayed by 1 day and the reaction was further improved . This unusual strategy has almost never been reported with other nitrile-converting strains . The resulting culture broth, containing methacrylamide as the inducer, beef extract as the nitrogen source and glucose as the carbon source, with methacrylamide added 24 h later, seemed to be most suitable . S-(+)-2-Phenylpropionic acid and R-(-)-2-phenylpropionamide were produced with yields of 48% (enantiomeric excess, 96%) and 42% (enantiomeric excess, 97%) respectively with no nitrile left after 3 h, or with yields of 52% and 39% (enantiomeric excess, 93% and 99%) respectively after 6 h. Appl Environ Microbiol, 2002 Feb, 68(2), 691 - 8 Chemostat approach for the directed evolution of biodesulfurization gain-of-function mutants; Arensdorf JJ et al.; Chemostat enrichment is a classical microbiological method that is well suited for use in directed-evolution strategies . We used a two-phase sulfur-limited chemostat to select for gain-of-function mutants with mutations in the biodesulfurization (Dsz) system of Rhodococcus erythropolis IGTS8, enriching for growth in the presence of organosulfur compounds that could not support growth of the wild-type strain . Mutations arose that allowed growth with octyl sulfide and 5-methylbenzothiophene as sole sulfur sources . An isolate from the evolved chemostat population was genetically characterized and found to contain mutations in two genes, dszA and dszC . A transversion (G to T) in dszC codon 261 resulted in a V261F mutation that was determined to be responsible for the 5-methylbenzothiophene gain-of-function phenotype . By using a modified RACHITT (random chimeragenesis on transient templates) method, mutant DszC proteins containing all possible amino acids at that position were generated, and this mutant set was assayed for the ability to metabolize 5-methylbenzothiophene, alkyl thiophenes, and dibenzothiophene . No mutant with further improvements in these catalytic activities was identified, but several clones lost all activity, confirming the importance of codon 261 for enzyme activity. Biotechnol Prog, 2002 Jan-Feb, 18(1), 88 - 93 Comparison of the emulsion characteristics of Rhodococcus erythropolis and Escherichia coli SOXC-5 cells expressing biodesulfurization genes; Borole AP et al.; Biodesulfurization of fuel oils is a two-phase (oil/water) process which may offer an interesting alternative to conventional hydrodesulfurization due to the mild operating conditions and reaction specificity afforded by the biocatalyst . For biodesulfurization to realize commercial success, a variety of process considerations must be addressed including reaction rate, emulsion formation and breakage, biocatalyst recovery, and both gas and liquid mass transport . This study evaluates emulsion formation and breakage using two biocatalysts with differing hydrophobic characteristics . A Gram-positive (Rhodococcus erythropolis) biocatalyst, expressing the complete 4S desulfurization pathway, and a Gram-negative biocatalyst (Escherichia coli), expressing only the gene for conversion of dibenzothiophene (DBT) to DBT sulfone, are compared relative to their ability to convert DBT and the ease of phase separation as well as biocatalyst recovery following desulfurization. Can J Microbiol, 2001 Dec, 47(12), 1107 - 15 DNA-based and culture-based characterization of a hydrocarbon-degrading consortium enriched from Arctic soil; Thomassin-Lacroix EJ et al.; A hydrocarbon-degrading consortium was enriched from fuel-contaminated soil from the northeastern tip of Ellesmere Island (82 degrees 30'N, 62 degrees 19'W) . The enrichment culture was grown on Jet A-1 fuel at 7 degrees C . Bacterial 16S RNA gene (rDNA) fragments were amplified by polymerase chain reaction (PCR) from members of the above consortium and cloned into a plasmid vector . Partial sequences (approximately 500 bp) were determined for 29 randomly selected rDNA clones . The majority of sequences were most similar to the corresponding rDNA sequences of Rhodococcus erythropolis (15 sequences), Sphingomonas spp . (six sequences), and Pseudomonas synxantha (four sequences) . Amplified ribosomal DNA restriction analysis confirmed that a larger set of 50 clones had frequencies of the three phylotypes similar to those above . Phylotype-specific PCR assays were developed and validated for the above three phylotypes . The consortium was plated and grown on Jet A-1 fuel vapors, and randomly selected isolated colonies were screened with the above PCR assays . Of 17 colonies, six matched the Rhodococcus phylotype, and three matched the Pseudomonas phylotype . A representative strain of each phylotype was physiologically characterized . Both isolates grew on alkanes at low temperature and had general characteristics consistent with their respective phylotypes . During growth of the consortium, the three phylotype populations were monitored by a most probable number PCR assay . All three phylotypes were detected, but their relative abundance was not consistent with that of the phylotypes in the clone library . The relative abundance of all three phylotypes changed substantially during long-term incubation of the consortium . The DNA-based approach used identified phylotypes consistently present in the consortium, but it failed to predict the relative abundance of their populations. Adv Biochem Eng Biotechnol, 2001, 73, 85 - 101 Metabolic engineering of indene bioconversion in Rhodococcus sp; Stafford DE et al.; We have applied the methodology of metabolic engineering in the investigation of the enzymatic bioreaction network in Rhodococcus sp . that catalyzes the bioconversion of indene to (2R)-indandiol suitable for the synthesis of cis-1-amino-2-indanol, a precursor of the HIV protease inhibitor, Crixivan . A chemostat with a novel indene air delivery system was developed to facilitate the study of steady state physiology of Rhodococcus sp . 124 . Prolonged cultivation of this organism in a continuous flow system led to the evolution of a mutant strain, designated KY1, with improved bioconversion properties, in particular a twofold increase in yield of (2R)-indandiol relative to 124 . Induction studies with both strains indicated that KY1 lacked a toluene-inducible dioxygenase activity present in 124 and responsible for the formation of undesired byproducts . Flux analysis of indene bioconversion in KY1 performed using steady state metabolite balancing and labeling with {14C}-tracers revealed that at least 94% of the indene is oxidized by a monooxygenase to indan oxide that is subsequently hydrolyzed to trans-(1R,2R)-indandiol and cis-(1S,2R)-indandiol . This analysis identified several targets in KY1 for increasing (2R)-indandiol product yield . Most promising among them is the selective hydrolysis of indan oxide to trans-(1R,2R)-indandiol through expression of an epoxide hydrolase or modification of culture conditions. J Infect Chemother, 2000 Dec, 6(4), 229 - 32 Pulmonary infection caused by Rhodococcus equi in HIV-infected patients: report of four patients from northern Thailand; Watanabe H et al.; We report four human immunodeficiency virus (HIV)-infected patients (3 men and one woman, average age, 34.3 years) with pulmonary infection (two with pneumonia and two with lung abscess) caused by Rhodococcus equi . These patients, who presented with fever and productive cough, were admitted to Nakornping Hospital in northern Thailand . Chest roentgenograms showed pulmonary infiltration and/or cavitary lesions . Their conditions were poor because of severe anemia, and transfusion was necessary in three of the four patients . Before culture results were available, the etiologic microorganisms identified in sputum smears were gram-positive and acid-fast coccobacilli . One of the four patients had a mixed infection with R . equi and Salmonella enteritidis . The mean CD4 lymphocyte count in the three tested patients was 10/mm3 (CD4/CD8 ratio = 0.057) . Four isolates of R . equi were sensitive to imipenem, minocycline, erythromycin, vancomycin, and ciprofloxacin (minimum inhibitory concentrations; MICs, <or=1.56 microg/ml), but resistant to most beta-lactam antibiotics . Two isolates were sensitive (MICs, 0.20 and 0.78 microg/ml) and two resistant (MICs 50 and >100 microg/ml) to rifampicin . Two patients were treated with erythromycin plus rifampicin, while the other two were treated with anti-tuberculous drugs . However, treatment was ineffective; three patients subsequently died because of respiratory failure, and one patient did not improve and was transferred to another hospital in her hometown. Arch Microbiol, 2002 Feb, 177(2), 159 - 66 Epub 2001 Nov 20. Expression of a functional NAD-reducing {NiFe} hydrogenase from the gram-positive Rhodococcus opacus in the gram-negative Ralstonia eutropha; Porthun A et al.; The actinomycete Rhodococcus opacus MR11 harbors a bidirectional NAD-reducing {NiFe} hydrogenase (SH) . This cytoplasmic enzyme is composed of two heterodimeric modules which catalyze distinct enzymatic activities . The hydrogenase moiety mediates H(2):benzyl viologen oxidoreductase activity and the FMN-containing diaphorase module displays NADH:benzyl viologen oxidoreductase activity . The SH of Rh . opacus resembles {NiFe} hydrogenases present in strains of the proteobacterium Ralstonia eutropha and in species of cyanobacteria . Heterologous expression of active {NiFe} hydrogenases failed in most cases due to protein-assisted maturation processes implicated in the assembly of the NiFe bimetallic site . This study reports on the construction of a recombinant plasmid harboring the four SH subunit genes hoxFUYH and the associated endopeptidase gene hoxW from Rh . opacus under the regime of the SH promoter from R . eutropha H16 . The resulting recombinant plasmid restored lithoautotrophic growth in a R . eutropha mutant impaired in H(2)-oxidizing ability . The SH of Rh . opacus was functionally active in R . eutropha and displayed the typical features described for its natural host . It readily dissociated in vitro into two active subforms . Dissociation was accompanied by the loss of the H(2)-dependent NAD-reducing activity, which was partially reconstituted by addition of 5 mM MgSO(4) and 0.5 mM NiCl(2) . Activity and stability of the SH from Rh . opacus were enhanced almost three-fold by co-overexpression of the SH-associated metal insertion genes hypA2B2F2 of R . eutropha . Under optimal conditions the heterologously expressed Rh . opacus SH catalyzed NAD-reduction at a specific activity of 1.7 units per mg protein, which is approximately 30% of the yield obtained for the R . eutropha SH . The results indicate that, despite an enormous phylogenetic distance of the two bacterial species, their SH proteins are highly related. J Bacteriol, 2002 Feb, 184(4), 1112 - 20 Chromosomal locus that affects pathogenicity of Rhodococcus fascians; Vereecke D et al.; The gram-positive plant pathogen Rhodococcus fascians provokes leafy gall formation on a wide range of plants through secretion of signal molecules that interfere with the hormone balance of the host . Crucial virulence genes are located on a linear plasmid, and their expression is tightly controlled . A mutant with a mutation in a chromosomal locus that affected virulence was isolated . The mutation was located in gene vicA, which encodes a malate synthase and is functional in the glyoxylate shunt of the Krebs cycle . VicA is required for efficient in planta growth in symptomatic, but not in normal, plant tissue, indicating that the metabolic requirement of the bacteria or the nutritional environment in plants or both change during the interaction . We propose that induced hyperplasia on plants represents specific niches for the causative organisms as a result of physiological alterations in the symptomatic tissue . Hence, such interaction could be referred to as metabolic habitat modification. Adv Space Res, 2001, 28(4), 707 - 12 Laboratory investigations of the survivability of bacteria in hypervelocity impacts; Burchell MJ et al.; It is now well established that material naturally moves around the Solar System, even from planetary surface to planetary surface . Accordingly, the idea that life is distributed throughout space and did not necessarily originate on the Earth but migrated here from elsewhere (Panspermia) is increasingly deemed worthy of consideration . If life arrived at the Earth from space, its relative speed will typically be of order many km s-1, and the resulting collision with the Earth and its atmosphere will be in the hypervelocity regime . A mechanism for the bacteria to survive such an impact is required . Therefore a programme of hypervelocity impacts in the laboratory at (4.5 +/- 0.6) km s-1 was carried out using bacteria (Rhodococcus) laden projectiles . After impacts on a variety of target materials (rock, glass and metal) attempts were made to culture Rhodococcus from the surface of the resulting craters and also from the target material ejected during crater formation . Control shots with clean projectiles yielded no evidence for Rhodococcus growth from any crater surface or ejecta . When projectiles doped with Rhodococcus were used no impact crater surface yielded colonies of Rhodococcus . However, for four shots of bacteria into rock (two on chalk and two on granite) the ejecta was afterwards found to give colonies of Rhodococcus . This was not true for shots onto glass . In addition, shots into aerogel (density 96 kg m-3) were also carried out (two with clean projectiles and two with projectiles with Rhodococcus) . This crudely simulated aero-capture in a planetary atmosphere . No evidence for Rhodococcus growth was found from the projectiles captured in the aerogel from any of the four shots . c2001 COSPAR . Published by Elsevier Science Ltd . All rights reserved. Arch Microbiol, 2001 Dec, 177(1), 20 - 8 Epub 2001 Oct 06. Preparative isolation of lipid inclusions from Rhodococcus opacus and Rhodococcus ruber and identification of granule-associated proteins; Kalscheuer R et al.; Triacylglycerol granules synthesized and accumulated by Rhodococcus opacus and Rhodococcus ruber were isolated by glycerol density gradient centrifugation . Whereas only one type of granule could be isolated from R . opacus, two types of granules with different specific densities were isolated from R . ruber . Both types of R . ruber granules showed a similar content of triacylglycerols and poly(3-hydroxybutyrate- co-3-hydroxyvalerate), but the protein profiles of both types were significantly different . The granules with the lower specific density were colorless; the granules with the higher specific density had a deep orange pigmentation . Solubilization studies revealed three different groups of granule-associated proteins: (1) unspecifically bound proteins, (2) relatively weakly associated proteins, and (3) proteins that resisted solubilization by treatment with 2 M NaCl, 2% (w/v) Triton X-114, 6 M guanidinium hydrochloride, up to 8% (w/v) SDS, and proteolytic digestion . The strong association of proteins of the last group suggested that these may play a specific role in the synthesis or mobilization of storage lipids or in the structure of the granules . The N-terminal amino acid sequences of the most tightly bound proteins were obtained . Proteins of low molecular weight with striking sequence similarity to the ribosomal protein L7 from various actinomycetes were always copurified with the granules. FEMS Microbiol Lett, 2002 Jan 2, 206(1), 75 - 9 The triazine hydrolase gene trzN from Nocardioides sp . strain C190: cloning and construction of gene-specific primers; Mulbry WW et al.; Using oligonucleotides derived from the N-terminal sequence of a triazine hydrolase from Nocardioides sp . strain C190, two DNA fragments containing trzN were cloned into Escherichia coli and their nucleotide sequences were determined . The 456-amino acid polypeptide predicted from the 1356-bp trzN ORF displayed significant similarity to triazine hydrolases from Pseudomonas and Rhodococcus isolates and belonged to the same amidohydrolase family . The trzN gene was flanked by two DNA sequences possessing 57 and 69% identity, respectively, at the protein level to Rhodococcus erythropolis sequences for a transposase and a transposase helper protein . Amplification primers specific to trzN were tested in soils inoculated with strain C190 . The results demonstrated that the primers were specific to trzN, and could detect populations at 10(8) cfu g(-1) soil using 250-mg soil samples. Mikrobiologiia, 2001 Nov-Dec, 70(6), 759 - 64 {Effect of the media salinity on destruction of petroleum oils by nocardioform bacteria}; Zviagintseva IS et al.; Oil degradation by cultures of Rhodococcus erythropolis and Dietzia maris was found to depend on the NaCl concentration in the medium . Optimal utilization of turbine oil by R . erythropolis and D . maris was observed at 0.5 and 2 to 5% NaCl concentration, respectively . Mineral oil and a mixture of paraffins (C14-C18) were utilized within a broader range of the medium salinity . As shown by fluorescent microscopy, D . maris colonies formed on the oil drop surface, whereas R . erythropolis cells penetrated the drops . The strains studied may populate various ecological niches in oil-containing ecosystems . They are promising for the development of microbial preparations for cleaning the environment from oil pollution. J Microbiol Methods, 2002 Feb, 48(2-3), 281 - 7 Fly-attracting volatiles produced by Rhodococcus fascians and Mycobacterium aurum isolated from myiatic lesions of sheep; Khoga JM et al.; Bacterial strains isolated from the healthy breech mucosa and myiatic wounds of ewes were tested for their volatile production as fly attractants towards Wohlfahrtia magnifica (Diptera: Sarcophagidae) . Cultures were studied as fly baits in field experiments, and strains performing with the best chemotropic effect were selected for further analysis . Static and dynamic headspace samples from shaken cultures were examined by gas chromatography-mass spectrometry (GC-MS) . Strains identified as Rhodococcus fascians and Mycobacterium aurum produced various volatile sulfur compounds and benzene, and proved to be the best fly attractants. Appl Environ Microbiol, 2002 Jan, 68(1), 166 - 72 Determination of key metabolites during biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine with Rhodococcus sp . strain DN22; Fournier D et al.; Rhodococcus sp . strain DN22 can convert hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to nitrite, but information on degradation products or the fate of carbon is not known . The present study describes aerobic biodegradation of RDX (175 microM) when used as an N source for strain DN22 . RDX was converted to nitrite (NO(2)(-)) (30%), nitrous oxide (N(2)O) (3.2%), ammonia (10%), and formaldehyde (HCHO) (27%), which later converted to carbon dioxide . In experiments with ring-labeled {(15)N}-RDX, gas chromatographic/mass spectrophotometric (GC/MS) analysis revealed N(2)O with two molecular mass ions: one at 44 Da, corresponding to (14)N(14)NO, and the second at 45 Da, corresponding to (15)N(14)NO . The nonlabeled N(2)O could be formed only from -NO(2), whereas the (15)N-labeled one was presumed to originate from a nitramine group ((15)N-(14)NO(2)) in RDX . Liquid chromatographic (LC)-MS electrospray analyses indicated the formation of a dead end product with a deprotonated molecular mass ion {M-H} at 118 Da . High-resolution MS indicated a molecular formula of C(2)H(5)N(3)O(3) . When the experiment was repeated with ring-labeled {(15)N}-RDX, the {M-H} appeared at 120 Da, indicating that two of the three N atoms in the metabolite originated from the ring in RDX . When {U-(14)C}-RDX was used in the experiment, 64% of the original radioactivity in RDX incorporated into the metabolite with a molecular weight (MW) of 119 (high-pressure LC/radioactivity) and 30% in (14)CO(2) (mineralization) after 4 days of incubation, suggesting that one of the carbon atoms in RDX was converted to CO(2) and the other two were incorporated in the ring cleavage product with an MW of 119 . Based on the above stoichiometry, we propose a degradation pathway for RDX based on initial denitration followed by ring cleavage to formaldehyde and the dead end product with an MW of 119. Microbiol Res, 2001, 156(4), 293 - 301 Studies on composition and stability of a large membered bacterial consortium degrading phenol; Ambujom S; A ten member microbial consortium (AS) consisting of eight phenol-degrading and two non-phenol-degrading strains of bacteria was developed and maintained in a fed-batch reactor by feeding 500 mg l(-1) phenol for four years at 28 +/- 3 degrees C . The consortium could degrade 99% of 500 mg l(-1) phenol after 24 hours incubation with a biomass increase of 2.6 x 10(7) to 4 x 10(12) CFU ml(-1) . Characterization of the members revealed that it consisted of 4 principal genera, Bacillus, Pseudomonas, Rhodococcus, Streptomyces and an unidentified bacterium . Phenol degradation by the mixed culture and Bacillus subtilis, an isolate from the consortium was compared using a range of phenol concentrations (400 to 700 mg l(-1)) and by mixing with either 160 mg l(-1) glucose or 50 mg l(-1) of 2,4-dichlorophenol in the medium . Simultaneous utilization of unrelated mixed substrates (glucose/2,4-dichlorophenol) by the consortium and Bacillus subtilis, indicated the diauxic growth pattern of the organisms . A unique characteristic of the members of the consortia was their ability to oxidize chloro aromatic compounds via meta pathway and methyl aromatic compounds via ortho cleavage pathway . The ability of a large membered microbial consortia to maintain its stability with respect to its composition and effectiveness in phenol degradation indicated its suitability for bioremediation applications. Environ Toxicol Chem, 2001 Dec, 20(12), 2681 - 9 Structure-specificity relationships for haloalkane dehalogenases; Damborsky J et al.; A structural analysis of the substrate specificity of hydrolytic dehalogenases originating from three different bacterial isolates has been performed using the multiple computer-automated structure evaluation methodology . This methodology identifies structural fragments in substrate molecules that either activate or deactivate biological processes . The analysis presented in this contribution is based on newly measured dehalogenation data combined with data from the literature (91 substrates) . The enzymes under study represent different specificity classes of haloalkane dehalogenases (haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, Rhodococcus erythropolis Y2, and Sphingomonas paucimobilis UT26) . Three sets of structural rules have been identified to explain their substrate specificity and to predict activity for untested substrates . Predictions of activity and inactivity based on the structural rules from this analysis were provided for those compounds that were not yet tested experimentally . Predictions were also made for the compounds with available experimental data not used for the model construction (i.e., the external validation set) . Correct predictions were obtained for 28 of 30 compounds in the validation set . Incorrect predictions were noted for two substrates outside the chemical domain of the set of compounds for which the structural rules were generated . A mechanistic interpretation of the structural rules generated provided a fundamental understanding of the structure-specificity relationships for the family of haloalkane dehalogenases. Mikrobiologiia, 2001 Sep-Oct, 70(5), 701 - 8 {Formation of artificial nitrogen-fixing symbiosis with rape (Brassica napus var . napus) plants in nonsterile soil}; Koval'skaia NIu et al.; The treatment of rape plants grown in nonsterile soil with 2,4-dichlorophenoxyacetic acid (auxin-like growth-promoting substance) or their inoculation with the bacterial association Micrococcus sp . + Rhodococcus sp . and/or with the mixed nitrogen-fixing culture Azotobacter nigricans + Bacillus sp . led to the formation of paranodules on the rape roots . The introduced bacteria were detected both in the intercellular space and inside the cells of the paranodules and the rape roots . The nitrogen-fixing activity of the paranodulated plants was two times higher than that of the inoculated plants lacking paranodules and five times higher than that of the control (i.e., not inoculated) plants . The paranodulation led to a 40% increase in the crop yield of rape plants and provided for a statistically significant increase in the total nitrogen as well as protein nitrogen contents of the plants. Am J Vet Res, 2001 Dec, 62(12), 1870 - 5 Pharmacokinetics of azithromycin and concentration in body fluids and bronchoalveolar cells in foals; Jacks S et al.; OBJECTIVE: To determine the pharmacokinetics of azithromycin and its concentration in body fluids and bronchoalveolar lavage cells in foals . ANIMALS: 6 healthy 6- to 10-week-old foals . PROCEDURE: Azithromycin (10 mg/kg of body weight) was administered to each foal via i.v . and intragastric (i.g.) routes in a crossover design . After the first i.g . dose, 4 additional i.g . doses were administered at 24-hour intervals . A microbiologic assay was used to measure azithromycin concentrations in serum, peritoneal fluid, synovial fluid, pulmonary epithelial lining fluid (PELF), and bronchoalveolar (BAL) cells . RESULTS: Azithromycin elimination half-life was 20.3 hours, body clearance was 10.4 ml/min x kg, and apparent volume of distribution at steady state was 18.6 L/kg . After i.g . administration, time to peak serum concentration was 1.8 hours and bioavailability was 56% . After repeated i.g . administration, peak serum concentration was 0.63 +/- 0.10 microg/ml . Peritoneal and synovial fluid concentrations were similar to serum concentrations . Bronchoalveolar cell and PELF concentrations were 15- to 170-fold and 1- to 16-fold higher than concurrent serum concentrations, respectively . No adverse reactions were detected after repeated i.g . administration . CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of pharmacokinetic values, minimum inhibitory concentrations of Rhodococcus equi isolates, and drug concentrations in PELF and bronchoalveolar cells, a single daily oral dose of 10 mg/kg may be appropriate for treatment of R . equi infections in foals . Persistence of high azithromycin concentrations in PELF and bronchoalveolar cells 48 hours after discontinuation of administration suggests that after 5 daily doses, oral administration at 48-hour intervals may be adequate. Appl Microbiol Biotechnol, 2001 Nov, 57(4), 460 - 6 Desulfurization and desulfonation: applications of sulfur-controlled gene expression in bacteria; Kertesz MA et al.; Inorganic sulfate is the preferred sulfur source for the growth of most microorganisms but, in its absence, many organosulfur compounds can be degraded microbially to provide sulfur . Desulfurization of dibenzothiophene (DBT) by Rhodococcus sp . and of aromatic sulfonates by Pseudomonas sp . has considerable biotechnological potential . Both these pathways require non-flavin-containing FMNH2-dependent monoxygenases (DszC/DszA and SsuD, respectively) . FMNH2 is provided from the freely diffusible FMNH2 pool in the cell, and is replenished by specific NAD(P)H:FMN oxidoreductases (DszD and SsuE) . Overexpression of the DszD FMN reductase in a heterologous system increases the efficiency of DBT desulfurization but is detrimental to cell growth at high levels . Expression of the sulfonatase that cleaves aromatic sulfonates (surfactants, dyes) is accompanied by synthesis of a thiol-specific antioxidant protein, which may protect the cell from superoxide radicals generated by autoxidation of the reduced flavin . Effective application of DBT desulfurization in the biodesulfurization of crude oil, and of arylsulfonate desulfonation in bioremediation, may require optimization of both flavin reductase levels and antioxidant protection systems within the cell. Antonie Van Leeuwenhoek, 2001 Oct, 80(2), 169 - 83 Physiology, biochemistry and taxonomy of deep-sea nitrile metabolising Rhodococcus strains; Heald SC et al.; A collection of nitrile-hydrolysing rhodococci was isolated from sediments sampled from a range of deep coastal, and abyssal and hadal trench sites in the NW Pacific Ocean, as part of our programme on the diversity of marine actinomycetes . Nitrile-hydrolysing strains were obtained by batch enrichments on nitrile substrates with or without dispersion and differential centrifugation pre-treatment of sediments, and were recovered from all of the depths sampled (approximately 1100-6500 m) . Two isolates obtained from the Ryukyu (5425 m) and Japan (6475 m) Trenches, and identified as strains of Rhodococcus erythropolis, were chosen for detailed study . Both of the deep-sea isolates grew at in situ temperature (4 degrees C), salinities (0-4% NaCl) and pressures (40-60 MPa), results that suggest, but do not prove, that they may be indigenous marine bacteria . However, the absence of culturable Thermoactinomyces points to little or no run off of terrestrial microbiota into these particular trench sediments . Nitrile-hydrolysis by these rhodococci was catalysed by a nitrile hydratase-amidase system . The hydratase accommodated aliphatic, aromatic and dinitrile substrates, and enabled growth to occur on a much wider range of nitriles than the only other reported marine nitrile-hydrolysing R . erythropolis which was isolated from coastal sediments . Also unlike the latter strain, the nitrile hydratases of the deep-sea rhodococci were constitutive . The possession of novel growth and enzyme activities on nitriles by these deep-sea R . erythropolis strains recommends their further development as industrial biocatalysts. FEMS Microbiol Lett, 2001 Dec 18, 205(2), 243 - 6 Random insertion mutagenesis of the intracellular pathogen Rhodococcus equi using transposomes; Mangan MW et al.; The identification of virulence factors in Rhodococcus equi has been severely hampered by the lack of a method for in vivo random insertion mutagenesis . This study reports the use of transposomes to generate random insertions of a gene conferring kanamycin resistance into the genome of R . equi ATCC 33701 . Southern hybridisation using the kanamycin resistance gene as probe showed that insertion of transposome is random . This was confirmed following nucleotide sequence analysis of the junction between the transposome and chromosomal DNA . The presence of a 9 bp duplication of the target sequence showed that random integration of the transposome was due to a bona fide Tn5 transposition event. FEMS Microbiol Lett, 2001 Dec 18, 205(2), 197 - 202 Unmarked gene deletion mutagenesis of kstD, encoding 3-ketosteroid Delta1-dehydrogenase, in Rhodococcus erythropolis SQ1 using sacB as counter-selectable marker; van der Geize R et al.; This paper reports the first method for the construction of unmarked gene deletion mutants in the genus Rhodococcus . Unmarked deletion of the kstD gene, encoding 3-ketosteroid Delta1-dehydrogenase (KSTD1) in Rhodococcus erythropolis SQ1, was achieved using the sacB counter-selection system . Conjugative mobilization of the mutagenic plasmid from Escherichia coli S17-1 to R . erythropolis strain SQ1 was used to avoid its random genomic integration . The kstD gene deletion mutant, designated strain RG1, still possessed about 10% of the KSTD enzyme activity of wild-type and was not affected in its ability to grow on the steroid substrates 4-androstene-3,17-dione (AD) and 9alpha-hydroxy-4-androstene-3,17-dione (9OHAD) . Biochemical evidence subsequently was obtained for the presence of a second KSTD enzyme (KSTD2) in R . erythropolis SQ1 . UV mutants of strain RG1 unable to grow on AD were isolated . One of these mutants, strain RG1-UV29, had lost all KSTD enzyme activity and was also unable to grow on 9OHAD . It stoichiometrically converted AD into 9OHAD in concentrations as high as 20 g x l(-1) . The two KSTD enzymes apparently both function in AD and 9OHAD catabolism . These isoenzymes have been inactivated in strain RG1 (KSTD1 negative) and strain RG1-UV29 (KSTD1 and KSTD2 negative), respectively. J Biol Chem, 2002 Mar 1, 277(9), 7051 - 8 Epub 2001 Dec 17. Barbiturase, a novel zinc-containing amidohydrolase involved in oxidative pyrimidine metabolism; Soong CL et al.; Barbiturase, which catalyzes the reversible amidohydrolysis of barbituric acid to ureidomalonic acid in the second step of oxidative pyrimidine degradation, was purified to homogeneity from Rhodococcus erythropolis JCM 3132 . The characteristics and gene organization of barbiturase suggested that it is a novel zinc-containing amidohydrolase that should be grouped into a new family of the amidohydrolases superfamily . The amino acid sequence of barbiturase exhibited 48% identity with that of herbicide atrazine-decomposing cyanuric acid amidohydrolase but exhibited no significant homology to other proteins, indicating that cyanuric acid amidohydrolase may have evolved from barbiturase . A putative uracil phosphoribosyltransferase gene was found upstream of the barbiturase gene, suggesting mutual interaction between pyrimidine biosynthesis and oxidative degradation . Metal analysis with an inductively coupled radiofrequency plasma spectrophotometer revealed that barbiturase contains approximately 4.4 mol of zinc per mol of enzyme . The homotetrameric enzyme had K(m) and V(max) values of 1.0 mm and 2.5 micromol/min/mg of protein, respectively, for barbituric acid . The enzyme specifically acted on barbituric acid, and dihydro-l-orotate, alloxan, and cyanuric acid competitively inhibited its activity . The full-length gene encoding the barbiturase (bar) was cloned and overexpressed in Escherichia coli . The kinetic parameters and physicochemical properties of the cloned enzyme were apparently similar to those of the wild-type. Biochem J, 2001 Dec 15, 360(Pt 3), 639 - 44 HstK, a cyanobacterial protein with both a serine/threonine kinase domain and a histidine kinase domain: implication for the mechanism of signal transduction; Phalip V et al.; Two distinct families of protein kinases are involved in signal transduction: Ser, Thr and Tyr kinases, which are predominantly found among eukaryotes, and His kinases, as part of bacterial two-component signalling systems . Genetic studies in Arabidopsis and Saccharomyces have demonstrated that bacterial-type two-component systems may act upstream of Ser/Thr kinases in the same signalling pathway, but how this coupling is accomplished remains unclear . In the present study, we report the characterization of a protein kinase, HstK, from the N(2)-fixing cyanobacterium Anabaena sp . PCC 7120, that possesses both a Ser/Thr kinase domain and a His kinase domain . Proteins with a structural architecture similar to that of HstK can be found in the eukaryote, Schizosaccharomyces pombe, and the bacterium, Rhodococcus sp . M5 . HstK was present in cells grown with NH(4)(+) or N(2) as the nitrogen source, but was absent in cells grown with NO(3)(-) . The hstK gene was inactivated and the mutant phenotype was characterized . The catalytic domain of the Ser/Thr kinase of HstK functionally replaced that of Hog1p, a well-characterized protein kinase required for the response to high osmolarity in the S . cerevisiae heterologous system . The unusual multidomain structure of HstK suggests that a two-component system could be directly coupled to Ser/Thr kinases in the same signal transduction pathway. J Vet Diagn Invest, 2001 Nov, 13(6), 489 - 94 Prevalence of virulent Rhodococcus equi in soil from five R . equi-endemic horse-breeding farms and restriction fragment length polymorphisms of virulence plasmids in isolates from soil and infected foals in Texas; Takai S et al.; Rhodococcus equi isolates (462) obtained from 64 soil samples collected on 5 R . equi-endemic horse-breeding farms and isolates from 100 infected foals in Texas were examined to determine the prevalence and genotypic diversity of virulence-associated plasmids . Isolates were tested for the presence of 15-17-kDa virulence-associated protein antigens (VapA) by immunoblotting and virulence-associated plasmids by PCR . Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases for estimation of size and comparison of polymorphisims . Rhodococcus equi were isolated from soil of all 5 farms; however, virulent R . equi were only isolated from 3 of the 5 farms and represented 18.8% (87 of 462) of total isolates . Of the 87 virulent soil isolates, 56 (64.5%) contained an 85-kb type I plasmid, 23 (26.4%) an 87-kb type I plasmid, 7 (8%) a newly defined 85-kb type III plasmid (Tx 43), and 1 (1.1%) a newly defined 85-kb type IV plasmid (Tx 47) . Of the 100 isolates from infected foals, 96 were virulent . Of the 96 virulent isolates, 51 (53.1%) contained an 85-kb type I plasmid, 39 (40.6%) an 87-kb type I plasmid, 4 (4.2%) an 85-kb type III plasmid (Tx 43), and 2 (2.1%) an 85-kb type IV plasmid (Tx 47) . There are at least 4 different R . equi virulence-associated plasmids in Texas, 2 of which have not previously been described . Based upon virulence plasmid typing, there is geographic diversity among isolates of R . equi from clinical and environmental samples on horse-breeding farms in Texas . There is not a strong correlation between the presence of virulent R . equi in farm soils and the R . equi disease status of those farms. Pharmacotherapy, 2001 Aug, 21(8), 998 - 1002 Rhodococcus equi pneumonia in a patient with human immunodeficiency virus: case report and review; Kwa AL et al.; Rhodococcus equi is a facultative, intracellular, gram-positive coccobacillus increasingly reported as an opportunistic pathogen in patients positive for human immunodeficiency virus (HIV) . An HIV-positive man developed R . equi pneumonia and sepsis . He failed to improve despite surgical drainage of localized infection and many empiric antibiotics . Time-kill studies of R . equi isolated from the patient were performed against various antimicrobial agents to optimize therapy . Levofloxacin seemed to offer excellent in vitro bactericidal activity . Antagonism was observed with certain antibiotic combinations . Our anecdotal case report suggests that fluoroquinolones such as levofloxacin may offer superior efficacy to standard therapy in rhodococcal infections; their clinical utility deserves further investigation . In view of potential antagonism, prospective susceptibility testing for various drugs and drug combinations should be considered when clinically indicated. Antimicrob Agents Chemother, 2001 Dec, 45(12), 3640 - 3 In vitro activities of a new ketolide, ABT-773, against multidrug-resistant gram-positive cocci; Singh KV et al.; The in vitro activities of ABT-773 were evaluated against 324 strains of gram-positive bacteria, including multidrug-resistant Staphylococcus spp . and Enterococcus spp . ABT-773 had lower MIC ranges, MICs at which 50% of isolates are inhibited (MIC(50)s), and MIC(90)s than erythromycin or clindamycin for almost all isolates tested . The MICs of ABT-773 were also lower than those of quinupristin-dalfopristin (Q-D) for methicillin-susceptible Staphylococcus aureus, Rhodococcus spp., and Streptococcus spp., while the MICs of Q-D were lower than those of ABT-773 for methicillin-resistant S . aureus and Enterococcus faecium, including vancomycin-resistant isolates. Annu Rev Phytopathol, 2001, 39, 27 - 52 Leafy gall formation by Rhodococcus fascians; Goethals K et al.; Rhodococcus fascians infects a wide range of plants, initiating the formation of leafy galls that consist of centers of shoot amplification and shoot growth inhibition . R . fascians is an epiphyte but it also can establish endophytic populations . Bacterial signals involved in symptom development initiate de novo cell division and shoot meristem formation in differentiated tissues . The R . fascians signals exert activities that are distinct from mere cytokinin effects, and the evidence points to a process that adopted cytokinin biosynthetic enzymes to form derivatives with unique activity . Genes implicated in leafy gall formation are located on a linear plasmid and are subject to a highly controlling, complex regulatory network, integrating autoregulatory compounds and environmental signals . Leafy galls are considered as centers with specific metabolic features, a niche where populations of R . fascians experience a selective advantage . Such "metabolic habitat modification" might be universal for gall-inducing bacteria. Biodegradation, 2001, 12(1), 39 - 47 Degradation of 1,3-dichloropropene by a soil bacterial consortium and Rhodococcus sp . AS2C isolated from the consortium; Ou LT et al.; A bacterial consortium capable of degrading the fumigant 1,3-D ((Z)- and (E)- 1,3-dichloropropene) was enriched from an enhanced soil . This mixed culture degraded (Z)- and (E)-1,3-D only in the presence of a suitable biodegradable organic substrate, such as tryptone, tryptophan, or alanine . After 8 months of subculturing at 2- to 3-week intervals, a strain of Rhodococcus sp . (AS2C) that was capable of degrading 1,3-D cometabolically in the presence of a suitable second substrate was isolated . (Z)-3-chloroallyl alcohol (3-CAA) and (Z)-3-chloroacrylic acid (3-CAAC), and (E)-3-CAA and (E)-3-CAAC were the metabolites of (Z)- and (E)- 1,3-D, respectively . (E)- 1,3-D was degraded faster than (Z)- 1,3-D by the strain AS2C and the consortium . AS2C also degraded (E)-3-CAA faster than (Z)-3-CAA . Isomerization of (E)- 1,3-D to (Z)- 1,3-D or the (Z) form to the (E) form did not occur. FEMS Microbiol Lett, 2001 Oct 16, 204(1), 205 - 11 Metabolism of anthracene by a Rhodococcus species; Dean-Ross D et al.; A Rhodococcus sp . isolated from contaminated river sediment was investigated to determine if the isolate could degrade high molecular mass polycyclic aromatic hydrocarbons . The Rhodococcus sp . was able to utilize anthracene (53%), phenanthrene (31%), pyrene (13%), and fluoranthene (5%) as sole source of carbon and energy, but not naphthalene or chrysene . In a study of the degradation of anthracene by a Rhodococcus sp., the identification of ring-fission products indicated at least two ring-cleavage pathways . One results in the production of 6,7-benzocoumarin, previously shown to be produced chemically from the product of meta cleavage of 1,2-dihydroxyanthracene, a pathway which has been well established in Gram-negative bacteria . The second is an ortho cleavage of 1,2-dihydroxyanthracene that produces 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, a dicarboxylic acid ring-fission product . This represents a novel metabolic pathway only identified in Gram-positive bacteria. Mol Microbiol, 2001 Oct, 42(1), 13 - 28 The att locus of Rhodococcus fascians strain D188 is essential for full virulence on tobacco through the production of an autoregulatory compound; Maes T et al.; The ability of Rhodococcus fascians strain D188 to provoke leafy gall formation on a variety of plant species is correlated with the linear plasmid pFiD188, on which different pathogenicity loci were identified . The att locus affects the severity of symptom development on tobacco, whereas the fas locus is essential for virulence . To gain insight into the function of the att locus, sequence and expression analyses were performed . The att locus contains nine open reading frames homologous to arginine and beta-lactam biosynthetic genes . att gene expression is transcriptionally induced by leafy gall extracts, but not by extracts of uninfected plants, and depends on the attR gene that encodes a LysR-type transcriptional regulator . The att locus proves to be essential for the formation of inducing factors (IFs) that are present in gall extracts . Because the induction of the fas locus also requires the presence of IFs in gall extracts, the att locus is proposed to play an important role in regulating the expression of the virulence loci of R . fascians. J Bacteriol, 2001 Nov, 183(22), 6551 - 7 Cloning of a genetically unstable cytochrome P-450 gene cluster