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Sample records for cavitada por rhodococcus

  1. Rhodococcus equi foal pneumonia.

    PubMed

    Cohen, Noah D

    2014-12-01

    Pneumonia caused by Rhodococcus equi is an important cause of disease and death in foals. This article reviews current knowledge of the epidemiology, clinical signs, diagnosis, treatment, prevention, and control of R equi pneumonia in foals. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Rhodococcus jostii: a home for Rhodococcus strain RHA1.

    PubMed

    Jones, Amanda L; Davies, Julian; Fukuda, Masao; Brown, Roselyn; Lim, Jesmine; Goodfellow, Michael

    2013-09-01

    The taxonomic position of Rhodococcus strain RHA1, an effective degrader of polychlorinated biphenyls with a large linear chromosome, was established using a polyphasic approach. The morphological and chemotaxonomic properties of the strain were typical of members of the genus Rhodococcus. The strain shared a high level of 16S rRNA sequence similarity (99.9 %) with the type strain of Rhodococcus jostii, a member of the Rhodococcus erythropolis subclade. The two strains shared a DNA:DNA relatedness value well above the cut-off point recommended for the circumscription of genomic species and had a broad range of phenotypic properties in common. The combination of genomic and phenotypic data show strain RHA1 to be a bona fide member of the species Rhodococcus jostii.

  3. Genomic analyses confirm close relatedness between Rhodococcus defluvii and Rhodococcus equi (Rhodococcus hoagii).

    PubMed

    Sangal, Vartul; Jones, Amanda L; Goodfellow, Michael; Hoskisson, Paul A; Kämpfer, Peter; Sutcliffe, Iain C

    2015-01-01

    Rhodococcus defluvii strain Ca11(T) was isolated from a bioreactor involved in extensive phosphorus removal. We have sequenced the whole genome of this strain, and our comparative genomic and phylogenetic analyses confirm its close relatedness with Rhodococcus equi (Rhodococcus hoagii) strains, which share >80 % of the gene content. The R. equi virulence plasmid is absent though most of the chromosomal R. equi virulence-associated genes are present in R. defluvii Ca11(T). These data suggest that although R. defluvii is an environmental organism, it has the potential to colonize animal hosts.

  4. Improved electroporation of Rhodococcus equi.

    PubMed

    Sekizaki, T; Tanoue, T; Osaki, M; Shimoji, Y; Tsubaki, S; Takai, S

    1998-02-01

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

  5. Rhodococcus equi Infections in Dogs.

    PubMed

    Bryan, L K; Clark, S D; Díaz-Delgado, J; Lawhon, S D; Edwards, J F

    2017-01-01

    Five cases of Rhodococcus equi infection in dogs were identified from 2003 to 2014. Three of the dogs had severe, internal lesions attributable to R. equi that have not been previously described: endophthalmitis, endocarditis, and suppurative pleuropneumonia. Isolates from 4 of the dogs were analyzed by polymerase chain reaction for Rhodococcus virulence-associated plasmid (vap) genes. One isolate was vapA-positive, 2 lacked a virulence plasmid, and 1 carried the novel vapN-associated plasmid (pVAPN) recently characterized in bovine isolates. The pVAPN plasmid has not been described in isolates cultured from companion animals. Four of the dogs either were receiving immunosuppressive drugs or had endocrinopathies. R. equi has the potential to cause significant infections in dogs, and immunocompromised animals should be considered at risk for infection.

  6. Cesium Accumulation and Growth Characteristics of Rhodococcus erythropolis CS98 and Rhodococcus sp. Strain CS402

    PubMed Central

    Tomioka, Noriko; Uchiyama, Hiroo; Yagi, Osami

    1994-01-01

    Growth and cesium accumulation characteristics of two cesium-accumulating bacteria isolated from soils were investigated. Rhodococcus erythropolis CS98 and Rhodococcus sp. strain CS402 accumulated high levels of cesium (approximately 690 and 380 μmol/g [dry weight] of cells or 92 and 52 mg/g [dry weight] of cells, respectively) after 24 h of incubation in the presence of 0.5 mM cesium. The optimum pH for cesium uptake by both Rhodococcus strains was 8.5. Rubidium and cesium assumed part of the role of potassium in the growth of both Rhodococcus strains. Potassium and rubidium inhibited cesium accumulation by these Rhodococcus strains. It is likely that both Rhodococcus strains accumulated cesium through a potassium transport system. PMID:16349312

  7. Cloning systems for Rhodococcus and related bacteria

    DOEpatents

    Finnerty, W.R.; Singer, M.E.

    1990-08-28

    A plasmid transformation system for Rhodococcus was developed using an Escherichia coli-Rhodococcus shuttle plasmid. Rhodococcus sp. H13-A contains three cryptic indigenous plasmids, designated pMVS100, pMVS200 and pMVS300, of 75, 19.5 and 13.4 kilobases (Kb), respectively. A 3.8 Kb restriction fragment of pMVS300 was cloned into pIJ30, a 6.3 Kb pBR322 derivative, containing the E. coli origin of replication (ori) and ampicillin resistance determinant (bla) as well as a Streptomyces gene for thiostrepton resistance, tsr. The resulting 10.1 Kb recombinant plasmid, designated pMVS301, was isolated from E. coli DH1 (pMVS301) and transformed into Rhodococcus sp. AS-50, a derivative of strain H13-A, by polyethylene glycol-assisted transformation of Rhodococcus protoplasts and selection for thiostrepton-resistant transformants. This strain was deposited with the ATCC on Feb. 1, 1988 and assigned ATCC 53719. The plasmid contains the Rhodococcus origin of replication. The plasmid and derivatives thereof can therefore be used to introduce nucleic acid sequences to and from Rhodococcus for subsequent expression and translation into protein. The isolated origin of replication can also be used in the construction of new vectors. 2 figs.

  8. Cloning systems for Rhodococcus and related bacteria

    DOEpatents

    Finnerty, William R.; Singer, Mary E.

    1990-01-01

    A plasmid transformation system for Rhodococcus was developed using an Escherichia coli-Rhodococcus shuttle plasmid. Rhodococcus sp. H13-A contains three cryptic indigenous plasmids, designated pMVS100, pMVS200 and pMVS300, of 75, 19.5 and 13.4 kilobases (Kb), respectively. A 3.8 Kb restriction fragment of pMVS300 was cloned into pIJ30, a 6.3 Kb pBR322 derivative, containing the E. coli origin of replication (ori) and ampicillin resistance determinant (bla) as well as a Streptomyces gene for thiostrepton resistance, tsr. The resulting 10.1 Kb recombinant plasmid, designated pMVS301, was isolated from E. coli DH1 (pMVS301) and transformed into Rhodococcus sp. AS-50, a derivative of strain H13-A, by polyethylene glycol-assisted transformation of Rhodococcus protoplasts and selection for thiostrepton-resistant transformants. This strain was deposited with the ATCC on Feb. 1, 1988 and assigned ATCC 53719. The plasmid contains the Rhodococcus origin of replication. The plasmid and derivatives thereof can therefore be used to introduce nucleic acid sequences to and from Rhodococcus for subsequent expression and translation into protein. The isolated origin of replication can also be used in the construction of new vectors.

  9. Antimicrobial resistance in Rhodococcus equi.

    PubMed

    Cisek, Agata A; Rzewuska, Magdalena; Witkowski, Lucjan; Binek, Marian

    2014-01-01

    Rhodococcus equi is an important etiologic agent of respiratory- and non-respiratory tract infections, diseases of animals and humans. Therapy includes the use of various group of chemotherapeutic agents, however resistance acquirement is quite common. To date there is no preferred treatment protocol for infections caused by isolates resistant to macrolides and rifampicin. The resistance acquirement is a result of many molecular mechanisms, some of which include alterations in the cell envelope composition and structure, activity of the efflux pumps, enzymatic destruction or inactivation of antibiotics, and changes in the target site. This paper contains an overview of antimicrobial susceptibility of R. equi, and explains the possible molecular mechanisms responsible for antimicrobial resistance in this particular microorganism.

  10. Rhodococcus defluvii sp. nov., isolated from wastewater of a bioreactor and formal proposal to reclassify [Corynebacterium hoagii] and Rhodococcus equi as Rhodococcus hoagii comb. nov.

    PubMed

    Kämpfer, P; Dott, W; Martin, K; Glaeser, S P

    2014-03-01

    A Gram-stain-positive, non-endospore-forming rod-shaped bacterium, strain Ca11(T), was isolated from a bioreactor with extensive phosphorus removal and was studied in detail for its taxonomic allocation. 16S rRNA gene sequence analysis revealed closest sequence similarity of the strain to type strains of [Corynebacterium hoagii] and Rhodococcus equi (98.9%), Rhodococcus koreensis and Rhodococcus wratislaviensis (both 98.4%), Rhodococcus opacus and Rhodococcus canchipurensis (both 98.0%) followed by Rhodococcus kunmingensis and Rhodococcus imtechensis (97.7%). Phylogenetic trees showed a distinct clustering of strain Ca11(T) with the type strains of [C. hoagii], R. equi, and R. kunmingensis separate to all other species of the genus Rhodococcus. The quinone system of strain Ca11(T) was composed of dihydrogenated menaquinones with 8 (major amount) as well as 7 and 6 isoprenoid units [MK-8(H2), MK-7(H2), MK-6(H2)]. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, one unknown phospholipid and an unidentified glycolipid. The fatty acid profile was similar to that reported for R. equi and contained major amounts of C16:0, C18:1ω9c and 10-methyl C18:0, supporting the allocation of the strain to the genus Rhodococcus. Physiological and biochemical characterization and DNA-DNA hybridization with type strains of the most closely related species allowed clear phenotypic and genotypic differentiation of the isolate. On the basis of these results, strain Ca11(T) ( = DSM 45893(T) = LMG 27563(T)) represents a novel species of the genus Rhodococcus, with the proposed name Rhodococcus defluvii sp. nov. In addition, a polyphasic taxonomic analysis of [Corynebacterium hoagii] DSM 20295(T) and Rhodococcus equi DSM 20307(T) indicated that the two strains belong to the same species, for which the name Rhodococcus hoagii comb. nov. takes priority, according to the Rules of the

  11. Construction of an Escherichia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp.

    PubMed Central

    Singer, M E; Finnerty, W R

    1988-01-01

    A plasmid transformation system for Rhodococcus sp. strain H13-A was developed by using an Escherichia coli-Rhodococcus shuttle plasmid constructed in this study. Rhodococcus sp. strain H13-A contains three cryptic indigenous plasmids, designated pMVS100, pMVS200, and pMVS300, of 75, 19.5, and 13.4 kilobases (kb), respectively. A 3.8-kb restriction fragment of pMVS300 was cloned into pIJ30, a 6.3-kb pBR322 derivative, containing the E. coli origin of replication (ori) and ampicillin resistance determinant (bla), as well as a Streptomyces gene for thiostrepton resistance, tsr. The resulting 10.1-kb recombinant plasmid, designated pMVS301, was isolated from E. coli DH1(pMVS301) and transformed into Rhodococcus sp. strain AS-50, a derivative of strain H13-A, by polyethylene glycol-assisted transformation of Rhodococcus protoplasts and selection for thiostrepton-resistant transformants. Thiostrepton-resistant transformants were also ampicillin resistant and were shown to contain pMVS301, which was subsequently isolated and transformed back into E. coli. The cloned 3.8-kb fragment of Rhodococcus DNA in pMVS301 contains a Rhodococcus origin of replication, since the hybrid plasmid was capable of replication in both genera. The plasmid was identical in E. coli and Rhodococcus transformants as determined by restriction analysis and was maintained as a stable, independent replicon in both organisms. Optimization of the transformation procedure resulted in transformation frequencies in the range of 10(5) transformants per micrograms of pMVS301 DNA in Rhodococcus sp. strain H13-A and derivative strains. The plasmid host range extends to strains of Rhodococcus erythropolis, R. globulerus, and R. equi, whereas stable transformants were not obtained with R. rhodochrous or with several coryneform bacteria tested as recipients. A restriction map demonstrated 14 unique restriction sites in pMVS301, some of which are potentially useful for molecular cloning in Rhodococcus spp. and

  12. Genetic Susceptibility to Rhodococcus equi.

    PubMed

    McQueen, C M; Dindot, S V; Foster, M J; Cohen, N D

    2015-01-01

    Rhodococcus equi pneumonia is a major cause of morbidity and mortality in neonatal foals. Much effort has been made to identify preventative measures and new treatments for R. equi with limited success. With a growing focus in the medical community on understanding the genetic basis of disease susceptibility, investigators have begun to evaluate the interaction of the genetics of the foal with R. equi. This review describes past efforts to understand the genetic basis underlying R. equi susceptibility and tolerance. It also highlights the genetic technology available to study horses and describes the use of this technology in investigating R. equi. This review provides readers with a foundational understanding of candidate gene approaches, single nucleotide polymorphism-based, and copy number variant-based genome-wide association studies, and next generation sequencing (both DNA and RNA). Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

  13. Construction of an Escherichia coli-rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp

    SciTech Connect

    Singer, M.E.V.; Finnerty, W.R.

    1988-02-01

    A plasmid transformation system for Rhodococcus sp. strain H13-A was developed by using an Escherichia coli-Rhodococcus shuttle plasmid constructed in this study. Rhodococcus sp. strain H13-A contains three cryptic indigenous plasmids, designated pMVS100, pMVS200, and pMVS300, of 75, 19.5, and 13.4 kilobases (kb), respectively. A 3.8-kb restriction fragment of pMVS300 was cloned into pIJ30, a 6.3-kb pBR322 derivative, containing the E. coli origin of replication (ori) and ampicillin resistance determinant (bla), as well as a Streptomyces gene for thiostrepton resistance, tsr. The resulting 10.1-kb recombinant plasmid, designated pMVS301, was isolated from E. coli DH1 (pMVS301) and transformed into Rhodococccus sp. strain AS-50, a derivative of strain H13-A. The cloned 3.8-kb fragment of Rhodococcus DNA in pMVS301 contains a Rhodococcus origin of replication, since the hybrid plasmid was capable of replication in both genera. The plasmid was identical in E. coli and Rhodococcus transformants as determined by restriction analysis and was maintained as a stable, independent replicon in both organisms. A restriction map demonstrated 14 unique restriction sites in pMVS301, some of which are potentially useful for molecular cloning in Rhodococcus spp. and other actinomycetes. This is the first report of plasmid transformation and of heterologous gene expression in a Rhodococcus sp.

  14. Rhodococcus cerastii sp. nov. and Rhodococcus trifolii sp. nov., two novel species isolated from leaf surfaces.

    PubMed

    Kämpfer, P; Wellner, S; Lohse, K; Lodders, N; Martin, K

    2013-03-01

    Two Gram-positive, non-endospore-forming rods, strains C5(T) and T8(T), were isolated from the phyllospheres of Cerastium holosteoides and Trifolium repens, respectively, and were studied in detail for their taxonomic position. 16S rRNA gene sequence analysis allocated both isolates clearly to the genus Rhodococcus. Isolate C5(T) was most closely related to Rhodococcus fascians and Rhodococcus yunnanensis, showing 99.2 % gene sequence similarity to both species. Strain T8(T) revealed the highest 16S rRNA gene sequence similarity to Rhodococcus corynebacterioides (98.8 %) and Rhodococcus kroppenstedtii (98.6 %). The quinone system of both strains was composed of dihydrogenated menaquinones with eight (major amount) as well as nine, seven and six isoprenoid units (MK-8H2, MK-9H2 MK-7H2 MK-6H2).The polar lipid profiles of strains C5(T) and T8(T) consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and one unknown phospholipid. Additionally, strain C5(T) contained one unknown glycolipid, and strain T8(T) three unknown aminolipids. The fatty acid profiles contained major amounts of C16 : 0, C18 : 1ω9c and 10-methyl C18 : 0, which supported the grouping of the two isolates in the genus Rhodococcus. Physiological/biochemical characterization and DNA-DNA hybridizations with the type strains of the most closely related species allowed a clear phenotypic and genotypic differentiation of both strains. For this reason, we propose strain C5(T) ( = LMG 26203(T)  = CCM 7906(T)) as the type strain of a novel species with the name Rhodococcus cerastii sp. nov., and strain T8(T) ( = LMG 26204(T)  = CCM 7905(T)) as the type strain of a second novel species with the name Rhodococcus trifolii sp. nov.

  15. Meningitis caused by Gordona aurantiaca (Rhodococcus aurantiacus).

    PubMed Central

    Prinz, G; Bán, E; Fekete, S; Szabó, Z

    1985-01-01

    In a case of hairy cell leukemia, Gordona aurantiaca (Rhodococcus aurantiacus) was isolated from cerebrospinal fluid as the pathogen responsible for lethal infection of the central nervous system. The pathogen had been isolated previously from one case of pulmonary infection process only. PMID:4044805

  16. Transfer of Tsukamurella wratislaviensis Goodfellow et a. 1995 to the genus Rhodococcus as Rhodococcus wratislaviensis comb. nov..

    PubMed

    Goodfellow, Michael; Chun, Jongsik; Stackebrandt, Erko; Kroppenstedt, Reiner M

    2002-05-01

    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..

  17. Mycotoxin-degradation profile of Rhodococcus strains.

    PubMed

    Cserháti, M; Kriszt, B; Krifaton, Cs; Szoboszlay, S; Háhn, J; Tóth, Sz; Nagy, I; Kukolya, J

    2013-08-16

    Mycotoxins are secondary fungal metabolites that may have mutagenic, carcinogenic, cytotoxic and endocrine disrupting effects. These substances frequently contaminate agricultural commodities despite efforts to prevent them, so successful detoxification tools are needed. The application of microorganisms to biodegrade mycotoxins is a novel strategy that shows potential for application in food and feed processing. In this study we investigated the mycotoxin degradation ability of thirty-two Rhodococcus strains on economically important mycotoxins: aflatoxin B1, zearalenone, fumonisin B1, T2 toxin and ochratoxin A, and monitored the safety of aflatoxin B1 and zearalenone degradation processes and degradation products using previously developed toxicity profiling methods. Moreover, experiments were performed to analyse multi-mycotoxin-degrading ability of the best toxin degrader/detoxifier strains on aflatoxin B1, zearalenone and T2 toxin mixtures. This enabled the safest and the most effective Rhodococcus strains to be selected, even for multi-mycotoxin degradation. We concluded that several Rhodococcus species are effective in the degradation of aromatic mycotoxins and their application in mycotoxin biodetoxification processes is a promising field of biotechnology.

  18. Rhodococcus enclensis sp. nov., a novel member of the genus Rhodococcus.

    PubMed

    Dastager, Syed G; Mawlankar, Rahul; Tang, Shan-Kun; Krishnamurthi, Srinivasan; Ramana, V Venkata; Joseph, Neeta; Shouche, Yogesh S

    2014-08-01

    A novel actinobacterial strain, designated, NIO-1009(T), was isolated from a marine sediment sample collected from Chorao Island, Goa, India. Phylogenetic analysis comparisons based on 16S rRNA gene sequences between strain NIO-1009(T) and other members of the genus Rhodococcus revealed that strain NIO-1009(T) had the closest sequence similarity to Rhodococcus kroppenstedtii DSM 44908(T) and Rhodococcus corynebacterioides DSM 20151(T) with 99.2 and 99.1%, respectively. Furthermore, DNA-DNA hybridization results showed that R. kroppenstedtii DSM 44908(T) and R. corynebacterioides DSM 20151(T) were 39.5 (3.0%) and 41.7 (2.0%) with strain NIO-1009(T), respectively, which were well below the 70% limit for any novel species proposal. Phylogenetically strain NIO-1009(T) forms a stable clade with and R. kroppenstedtii DSM 44908(T) and R. corynebacterioides DSM 20151(T) with 100% bootstrap values. Strain NIO-1009(T) contained meso-diaminopimelic acid as the diagnostic diamino acid and galactose and arabinose as the cell wall sugars. The major fatty acids were C(16 : 0), C(18 : 1)ω9c, C(16 : 1)(ω6c and/or ω7c) and 10-methyl C(18 : 0). The only menaquinone detected was MK-8(H2), while the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and one unknown phospholipid. The G+C content of the genomic DNA was 66.9 mol%. The phenotypic and genotypic data showed that strain NIO-1009(T) warrants recognition as a novel species of the genus Rhodococcus for which the name Rhodococcus enclensis sp. nov., is proposed; the type strain is NIO-1009(T) ( = NCIM 5452(T) = DSM 45688(T)).

  19. Treatment of Infections Caused by Rhodococcus equi.

    PubMed

    Giguère, Steeve

    2017-04-01

    Pneumonia caused by Rhodococcus equi remains an important cause of disease and death in foals. The combination of a macrolide (erythromycin, azithromycin, or clarithromycin) with rifampin remains the recommended therapy for foals with clinical signs of infection caused by R equi. Most foals with small, subclinical ultrasonographic pulmonary lesions associated with R equi recover without therapy, and administration of antimicrobial agents to these subclinically affected foals does not hasten lesion resolution relative to administration of a placebo. Resistance to macrolides and rifampin in isolates of R equi is increasing. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Trehalolipid biosurfactants from nonpathogenic Rhodococcus actinobacteria with diverse immunomodulatory activities.

    PubMed

    Kuyukina, Maria S; Ivshina, Irena B; Baeva, Tatiana A; Kochina, Olesia A; Gein, Sergey V; Chereshnev, Valery A

    2015-12-25

    Actinobacteria of the genus Rhodococcus produce trehalolipid biosurfactants with versatile biochemical properties and low toxicity. In recent years, these biosurfactants are increasingly studied as possible biomedical agents with expressed immunological activities. Applications of trehalolipids from Rhodococcus, predominantly cell-bound, in biomedicine are also attractive because their cost drawback could be less significant for high-value products. The review summarizes recent findings in immunomodulatory activities of trehalolipid biosurfactants from nonpathogenic Rhodococcus and related actinobacteria and compares their biomedical potential with well-known immunomodifying properties of trehalose dimycolates from Mycobacterium tuberculosis. Molecular mechanisms of trehalolipid interactions with immunocompetent cells are also discussed.

  1. Draft genome sequence of Rhodococcus rhodochrous strain ATCC 17895

    PubMed Central

    Chen, Bi-Shuang; Otten, Linda G.; Resch, Verena; Muyzer, Gerard; Hanefeld, Ulf

    2013-01-01

    Rhodococcus rhodochrous ATCC 17895 possesses an array of mono- and dioxygenases, as well as hydratases, which makes it an interesting organism for biocatalysis. R. rhodochrous is a Gram-positive aerobic bacterium with a rod-like morphology. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 6,869,887 bp long genome contains 6,609 protein-coding genes and 53 RNA genes. Based on small subunit rRNA analysis, the strain is more likely to be a strain of Rhodococcus erythropolis rather than Rhodococcus rhodochrous. PMID:24501654

  2. Use of whole genome sequences to develop a molecular phylogenetic framework for Rhodococcus fascians and the Rhodococcus genus

    PubMed Central

    Creason, Allison L.; Davis, Edward W.; Putnam, Melodie L.; Vandeputte, Olivier M.; Chang, Jeff H.

    2014-01-01

    The accurate diagnosis of diseases caused by pathogenic bacteria requires a stable species classification. Rhodococcus fascians is the only documented member of its ill-defined genus that is capable of causing disease on a wide range of agriculturally important plants. Comparisons of genome sequences generated from isolates of Rhodococcus associated with diseased plants revealed a level of genetic diversity consistent with them representing multiple species. To test this, we generated a tree based on more than 1700 homologous sequences from plant-associated isolates of Rhodococcus, and obtained support from additional approaches that measure and cluster based on genome similarities. Results were consistent in supporting the definition of new Rhodococcus species within clades containing phytopathogenic members. We also used the genome sequences, along with other rhodococcal genome sequences to construct a molecular phylogenetic tree as a framework for resolving the Rhodococcus genus. Results indicated that Rhodococcus has the potential for having 20 species and also confirmed a need to revisit the taxonomic groupings within Rhodococcus. PMID:25237311

  3. Use of whole genome sequences to develop a molecular phylogenetic framework for Rhodococcus fascians and the Rhodococcus genus.

    PubMed

    Creason, Allison L; Davis, Edward W; Putnam, Melodie L; Vandeputte, Olivier M; Chang, Jeff H

    2014-01-01

    The accurate diagnosis of diseases caused by pathogenic bacteria requires a stable species classification. Rhodococcus fascians is the only documented member of its ill-defined genus that is capable of causing disease on a wide range of agriculturally important plants. Comparisons of genome sequences generated from isolates of Rhodococcus associated with diseased plants revealed a level of genetic diversity consistent with them representing multiple species. To test this, we generated a tree based on more than 1700 homologous sequences from plant-associated isolates of Rhodococcus, and obtained support from additional approaches that measure and cluster based on genome similarities. Results were consistent in supporting the definition of new Rhodococcus species within clades containing phytopathogenic members. We also used the genome sequences, along with other rhodococcal genome sequences to construct a molecular phylogenetic tree as a framework for resolving the Rhodococcus genus. Results indicated that Rhodococcus has the potential for having 20 species and also confirmed a need to revisit the taxonomic groupings within Rhodococcus.

  4. [Periprosthetic joint infection caused by Rhodococcus equi. Case report].

    PubMed

    Sallai, Imre; Péterfy, Nóra; Sanatkhani, Mohammad; Bejek, Zoltán; Antal, Imre; Prinz, Gyula; Kristóf, Katalin; Skaliczki, Gábor

    2017-07-01

    Rhodococcus equi is a rare pathogen in humans causing infections mostly in immunocompromised hosts. We present the first case of periprosthetic joint infection caused by Rhodococcus equi. An 88-year-old male patient was referred to our clinic with a history of fever and right hip pain. The patient had multiple hip surgeries including total joint arthroplasty and revision for aseptic loosening on the right side. He was immunocompetent, but his additional medical history was remarkable for diabetes mellitus, diabetic nephropathy and stroke with hemiplegia resulting in immobilization. Radiography showed stable components, joint aspirate yielded Rhodococcus equi. Irrigation and debridement was proposed, but the patient refused any surgical intervention. Therefore antibiotic therapy was administered. At the last follow-up the patient is free of complaints but the C-reactive protein level is still elevated. This case illustrates the possible role of Rhodococcus equi in medical device-associated infections. Orv Hetil. 2017; 158(27): 1071-1074.

  5. Rhodococcus kyotonensis sp. nov., a novel actinomycete isolated from soil.

    PubMed

    Li, Bing; Furihata, Keiko; Ding, Lin-Xian; Yokota, Akira

    2007-09-01

    A polyphasic study was undertaken to establish the taxonomic position of an isolate, strain DS472(T), from soil in Kyoto, Japan. Phylogenetic analysis, based on the 16S rRNA gene sequences, revealed that this strain constitutes a new subline within the genus Rhodococcus, with Rhodococcus yunnanensis YIM 70056(T) and Rhodococcus fascians DSM 20669(T) as its nearest phylogenetic neighbours (98.2 and 97.8 % sequence similarity, respectively). DNA-DNA hybridization experiments revealed 36 and 29 % relatedness between the isolate and its phylogenetic relatives, R. yunnanensis and R. fascians, respectively. Chemotaxonomic characteristics, including the major quinone MK-8(H(2)), predominant fatty acids C(16 : 0), C(18 : 1)omega9c and 10-methyl C(18 : 0), the presence of cell-wall chemotype IV and mycolic acids, were consistent with the properties of members of the genus Rhodococcus. The DNA G+C content was 64.5 mol%. On the basis of both phenotypic and genotypic evidence, strain DS472(T) represents a novel species of the genus Rhodococcus, for which the name Rhodococcus kyotonensis sp. nov. is proposed. The type strain is strain DS472(T) (=IAM 15415(T)=CCTCC AB206088(T)).

  6. Leafy gall formation by Rhodococcus fascians.

    PubMed

    Goethals, K; Vereecke, D; Jaziri, M; Van Montagu, M; Holsters, M

    2001-01-01

    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.

  7. Glycogenformation by Rhodococcus species and the effect of inhibition of lipid biosynthesis on glycogen accumulation in Rhodococcus opacus PD630.

    PubMed

    Hernández, Martín A; Alvarez, Héctor M

    2010-11-01

    Members of the genus Rhodococcus were investigated for their ability to produce glycogen during cultivation on gluconate or glucose. Strains belonging to Rhodococcus ruber, Rhodococcus opacus, Rhodococcus fascians, Rhodococcus erythropolis and Rhodococcus equi were able to produce glycogen up to 0.2–5.6% of cellular dry weight (CDW). The glycogen content varied from 0.8% to 3.2% of CDW in cells of R. opacus PD630, which is a well-known oleaginous bacterium, during the exponential growth phase, when cultivated on diverse carbon sources. Maltose and pyruvate promoted glycogen accumulation by cells of strain PD630 to a greater extent than glucose, gluconate, lactose, sucrose or acetate. This strain was able to produce triacylglycerols, polyhydroxyalkanoates and glycogen as storage compounds during growth on gluconate, although triacylglycerols were always the main product under the conditions of this study. Cerulenin, an inhibitor of de novo fatty acid synthesis, inhibited the accumulation of triacylglycerols from gluconate and increased the content of polyhydroxyalkanoates (from 2.0% to 4.2%, CDW) and glycogen (from 0.1% to 3.0%, CDW). An increase of the polyhydroxyalkanoates and glycogen content was also observed in two mutants of R. opacus PD630, which produced reduced amounts of triacylglycerols during cultivation of cells on gluconate.

  8. Study of lysozyme resistance in Rhodococcus equi.

    PubMed

    Hébert, Laurent; Bidaud, Pauline; Goux, Didier; Benachour, Abdellah; Laugier, Claire; Petry, Sandrine

    2014-03-01

    Lysozyme is an important and widespread component of the innate immune response that constitutes the first line of defense against bacterial pathogens. The bactericidal effect of this enzyme relies on its capacity to hydrolyze the bacterial cell wall and also on a nonenzymatic mechanism involving its cationic antimicrobial peptide (CAMP) properties, which leads to membrane permeabilization. In this paper, we report our findings on the lysozyme resistance ability of Rhodococcus equi, a pulmonary pathogen of young foals and, more recently, of immunocompromised patients, whose pathogenic capacity is conferred by a large virulence plasmid. Our results show that (i) R. equi can be considered to be moderately resistant to lysozyme, (ii) the activity of lysozyme largely depends on its muramidase action rather than on its CAMP activity, and (iii) the virulence plasmid confers part of its lysozyme resistance capacity to R. equi. This study is the first one to demonstrate the influence of the virulence plasmid on the stress resistance capacity of R. equi and improves our understanding of the mechanisms enabling R. equi to resist the host defenses.

  9. Rhodococcus equi: an animal and human pathogen.

    PubMed Central

    Prescott, J F

    1991-01-01

    Recent isolations of Rhodococcus equi from cavitatory pulmonary disease in patients with AIDS have aroused interest among medical microbiologists in this unusual organism. Earlier isolations from humans had also been in immunosuppressed patients following hemolymphatic tumors or renal transplantation. This organism has been recognized for many years as a cause of a serious pyogranulomatous pneumonia of young foals and is occasionally isolated from granulomatous lesions in several other species, in some cases following immunosuppression. The last decade has seen many advances in understanding of the epidemiology, pathogenesis, diagnosis, treatment, and immunity to infection in foals. The particular susceptibility of the foal is not understood but can be explained in part by a combination of heavy challenge through the respiratory route coinciding with declining maternally derived antibody in the absence of fully competent foal cellular immune mechanisms. R. equi is largely a soil organism but is widespread in the feces of herbivores. Its growth in soil is considerably improved by simple nutrients it obtains from herbivore manure. About one-third of human patients who have developed R. equi infections had contact in some way with herbivores or their manure. Others may have acquired infection from contact with soil or wild bird manure. R. equi is an intracellular parasite, which explains the typical pyogranulomatous nature of R. equi infections, the predisposition to infection in human patients with defective cell-mediated immune mechanisms, and the efficacy of antimicrobial drugs that penetrate phagocytic cells. Images PMID:2004346

  10. Rhodococcus Bacteremia in Cancer Patients Is Mostly Catheter Related and Associated with Biofilm Formation

    PubMed Central

    Al Akhrass, Fadi; Al Wohoush, Iba; Chaftari, Anne-Marie; Reitzel, Ruth; Jiang, Ying; Ghannoum, Mahmoud; Tarrand, Jeffrey; Hachem, Ray; Raad, Issam

    2012-01-01

    Rhodococcus is an emerging cause of opportunistic infection in immunocompromised patients, most commonly causing cavitary pneumonia. It has rarely been reported as a cause of isolated bacteremia. However, the relationship between bacteremia and central venous catheter is unknown. Between 2002 and 2010, the characteristics and outcomes of seventeen cancer patients with Rhodococcus bacteremia and indwelling central venous catheters were evaluated. Rhodococcus bacteremias were for the most part (94%) central line-associated bloodstream infection (CLABSI). Most of the bacteremia isolates were Rhodococcus equi (82%). Rhodococcus isolates formed heavy microbial biofilm on the surface of polyurethane catheters, which was reduced completely or partially by antimicrobial lock solution. All CLABSI patients had successful response to catheter removal and antimicrobial therapy. Rhodococcus species should be added to the list of biofilm forming organisms in immunocompromised hosts and most of the Rhodococcus bacteremias in cancer patients are central line associated. PMID:22427914

  11. Unusual Extrapulmonary Rhodococcus Equi Infection in a Kidney Transplant Patient.

    PubMed

    Varotti, Giovanni; Barabani, Caterina; Dodi, Ferdinando; Bertocchi, Massimo; Mondello, Rosalia; Cupo, Pierpaolo; Santori, Gregorio; Palombo, Domenico; Fontana, Iris

    2016-12-01

    Rhodococcus equi is a well-recognized pathogen in veterinary medicine that can also affect immuno-compromised human subjects. The most common clinical features in humans include necrotizing pneumonia with subacute pulmonary disease, progressive cough, chest pain and fever. We report a case of a 49-year-old kidney transplant patient who developed a Rhodococcus equi infection characterized by multiple abscesses of the soft tissues and muscles without any respiratory manifestation. Combining specific antibiotic therapy and surgical management of the abscesses without immunosuppression discontinuation led to a complete recovery of both patient and graft.

  12. Rhodococcus pedocola sp. nov. and Rhodococcus humicola sp. nov., two antibiotic-producing actinomycetes isolated from soil.

    PubMed

    Nguyen, Tuan Manh; Kim, Jaisoo

    2016-06-01

    Two novel actinobacterial strains, UC12T and UC33T, were isolated from forest topsoil in Suwon, Gyeonggi-Do, South Korea. Comparative analysis of nearly full-length 16S rRNA gene sequences of UC12T and UC33T revealed close pairwise similarity with species of the genus Rhodococcus, and the UC12T and UC33T sequences were most closely related to Rhodococcus canchipurensis MBRL 353T (98.91 % 16S rRNA gene sequence similarity) and Rhodococcus triatomae IMMIB RIV-085T (97.71 %), respectively. DNA-DNA hybridization showed 33.05-35.60 % genomic similarity between strains UC12T and UC33T, while strain UC12T shared DNA-DNA relatedness values of 32.71-41.29 % with the closest species of the genus Rhodococcus and strain UC33T shared 29.12-37.91 % genomic relatedness with the closest species of the genus Rhodococcus. Both strains showed similar chemotaxonomic characteristics. The major fatty acids were C16 : 0, summed feature 3, C18 : 1ω9c and C18 : 0 10-methyl. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. The major quinone derived was MK-8(H2). The cell-wall peptidoglycan contained meso-diaminopimelic acid, and galactose, glucose, arabinose and ribose were detected in whole cells. Mycolic acids were detected. The DNA G+C content of strains UC12T and UC33T was 72.7 mol% and 68.8 mol%, respectively. Both strains produced antibiotic(s) that inhibited bacterial pathogens but not fungi. Based on the physiological, biochemical and genotypic features and the DNA-DNA hybridization between the isolates and type strains of closely related species, we propose that these bacteria be classified as novel species of the genus Rhodococcus with the names Rhodococcus pedocola sp. nov. (type strain UC12T=KACC 18499T=NBRC 111580T) and Rhodococcus humicola sp. nov. (type strain UC33T=KACC 18500T=NBRC 111581T).

  13. Can whole genome analysis refine the taxonomy of the genus Rhodococcus?

    PubMed

    Gürtler, Volker; Mayall, Barrie C; Seviour, Robert

    2004-06-01

    The current systematics of the genus Rhodococcus is unclear, partly because many members were originally included before the application of a polyphasic taxonomic approach, central to which is the acquisition of 16S rRNA sequence data. This has resulted in the reclassification and description of many new species. Hence, the literature is replete with new species names that have not been brought together in an organized and easily interpreted form. This taxonomic confusion has been compounded by assigning many xenobiotic degrading isolates with phylogenetic positions but without formal taxonomic descriptions. In order to provide a framework for a taxonomic approach based on multiple genetic loci, a survey was undertaken of the known genome characteristics of members of the genus Rhodococcus including: (i) genetics of cell envelope biosynthesis; (ii) virulence genes; (iii) gene clusters involved in metabolic degradation and industrially relevant pathways; (iv) genetic analysis tools; (v) rapid identification of bacteria including rhodococci with specific gene RFLPs; (vi) genomic organization of rrn operons. Genes encoding virulence factors have been characterized for Rhodococcus equi and Rhodococcus fascians. Based on peptide signature comparisons deduced from gene sequences for cytochrome P-450, mono- and dioxygenases, alkane degradation, nitrile metabolism, proteasomes and desulfurization, phylogenetic relationships can be deduced for Rhodococcus erythropolis, Rhodococcus globerulus, Rhodococcus ruber and a number of undesignated Rhodococcus spp. that may distinguish the genus Rhodococcus into two further genera. The linear genome topologies that exist in some Rhodococcus species may alter a previously proposed model for the analysis of genomic fingerprinting techniques used in bacterial systematics.

  14. Draft Genome Sequence of Subantarctic Rhodococcus sp. Strain 1139

    PubMed Central

    Baker, Anthony L.; Charleston, Michael A.; Britz, Margaret L.

    2017-01-01

    ABSTRACT The draft genome sequence of subantarctic Rhodococcus sp. strain 1139 is reported here. The genome size is 7.04 Mb with high G+C content (62.3%) and it contains a large number of genes involved in lipid synthesis. This lipid synthesis system is characteristic of oleaginous Actinobacteria, which are of interest for biofuel production. PMID:28385836

  15. Draft Genome Sequence of Rhodococcus rhodochrous Strain ATCC 21198

    SciTech Connect

    Shields-Menard, Sara A.; Brown, Steven D; Klingeman, Dawn Marie; Indest, Karl; Hancock, Dawn; Wewalwela, Jayani; French, Todd; Donaldson, Janet

    2014-01-01

    Rhodococcus rhodochrous is a Gram-positive red-pigmented bacterium commonly found in the soil. The draft genome sequence for R. rhodochrous strain ATCC 21198 is presented here to provide genetic data for a better understanding of its lipid-accumulating capabilities.

  16. Draft Genome Sequence of Rhodococcus sp. Strain 66b

    PubMed Central

    Myers, Cindy A.; O’Sullivan, Cathryn A.; Roper, Margaret M.

    2017-01-01

    ABSTRACT We report here the draft genome sequence and annotation of Rhodococcus sp. strain 66b isolated from the soil of southwest Western Australia. This strain exhibits a range of bioactivities, including plant growth promotion, biosurfactant production, and wax degradation. Whole-genome sequencing was conducted to uncover the underlying mechanisms. PMID:28546474

  17. Acute osteomyelitis caused by Rhodococcus equi in an immunocompetent child.

    PubMed

    Sistla, Sujatha; Karthikeyan, Sivasangeetha; Biswas, Rakhi; Parija, Subhash Chandra; Patro, Dilip Kumar

    2009-01-01

    Rhodococcus equi is an unusual pathogen causing infections mostly in immunocompromised patients, particularly in those with human immunodeficiency virus (HIV). It has rarely been reported to affect immunocompetent hosts, where it usually presents as an isolated extrapulmonary lesion. We report a case of osteomyelitis caused by this organism in an immunocompetent host.

  18. Gene Cluster Encoding Cholate Catabolism in Rhodococcus spp.

    PubMed Central

    Wilbrink, Maarten H.; Casabon, Israël; Stewart, Gordon R.; Liu, Jie; van der Geize, Robert; Eltis, Lindsay D.

    2012-01-01

    Bile acids are highly abundant steroids with important functions in vertebrate digestion. Their catabolism by bacteria is an important component of the carbon cycle, contributes to gut ecology, and has potential commercial applications. We found that Rhodococcus jostii RHA1 grows well on cholate, as well as on its conjugates, taurocholate and glycocholate. The transcriptome of RHA1 growing on cholate revealed 39 genes upregulated on cholate, occurring in a single gene cluster. Reverse transcriptase quantitative PCR confirmed that selected genes in the cluster were upregulated 10-fold on cholate versus on cholesterol. One of these genes, kshA3, encoding a putative 3-ketosteroid-9α-hydroxylase, was deleted and found essential for growth on cholate. Two coenzyme A (CoA) synthetases encoded in the cluster, CasG and CasI, were heterologously expressed. CasG was shown to transform cholate to cholyl-CoA, thus initiating side chain degradation. CasI was shown to form CoA derivatives of steroids with isopropanoyl side chains, likely occurring as degradation intermediates. Orthologous gene clusters were identified in all available Rhodococcus genomes, as well as that of Thermomonospora curvata. Moreover, Rhodococcus equi 103S, Rhodococcus ruber Chol-4 and Rhodococcus erythropolis SQ1 each grew on cholate. In contrast, several mycolic acid bacteria lacking the gene cluster were unable to grow on cholate. Our results demonstrate that the above-mentioned gene cluster encodes cholate catabolism and is distinct from a more widely occurring gene cluster encoding cholesterol catabolism. PMID:23024343

  19. Complete Genome and Plasmid Sequences for Rhodococcus fascians D188 and Draft Sequences for Rhodococcus Isolates PBTS 1 and PBTS 2

    PubMed Central

    Stamler, Rio A.; Vereecke, Danny; Zhang, Yucheng; Schilkey, Faye; Devitt, Nico

    2016-01-01

    Rhodococcus fascians, a phytopathogen that alters plant development, inflicts significant losses in plant production around the world. We report here the complete genome sequence of R. fascians D188, a well-characterized model isolate, and Rhodococcus species PBTS (pistachio bushy top syndrome) 1 and 2, which were shown to be responsible for a disease outbreak in pistachios. PMID:27284129

  20. Complete Genome and Plasmid Sequences for Rhodococcus fascians D188 and Draft Sequences for Rhodococcus Isolates PBTS 1 and PBTS 2.

    PubMed

    Stamler, Rio A; Vereecke, Danny; Zhang, Yucheng; Schilkey, Faye; Devitt, Nico; Randall, Jennifer J

    2016-06-09

    Rhodococcus fascians, a phytopathogen that alters plant development, inflicts significant losses in plant production around the world. We report here the complete genome sequence of R. fascians D188, a well-characterized model isolate, and Rhodococcus species PBTS (pistachio bushy top syndrome) 1 and 2, which were shown to be responsible for a disease outbreak in pistachios.

  1. [Adaptation of coimmobilized Rhodococcus cells to oil hydrocarbons in a column bioreactor].

    PubMed

    Serebrennikova, M K; Kuiukina, M S; Krivoruchko, A V; Ivshina, I B

    2014-01-01

    The possible adaptation of the association of Rhodococcus ruber and Rhodococcus opacus strains immobilized on modified sawdust to oil hydrocarbons in a column bioreactor was investigated. In the bioreactor, the bacterial population showed higher hydrocarbon and antibiotic resistance accompanied by the changes in cell surface properties (hydrophobicity, electrokinetic potential) and in the content of cellular lipids and biosurfactants. The possibility of using adapted Rhodococcus strains for the purification of oil-polluted water in the bioreactor was demonstrated.

  2. Molecular biological enhancement of coal biodesulfurization. [Rhodococcus rhodochrous

    SciTech Connect

    Kilbane, J.J.; Bielaga, B.A.

    1990-07-01

    The overall objective of this project is to sue molecular genetics to develop strains of bacteria with enhanced ability to remove sulfur from coal and to obtain data that will allow the performance and economics of a coal biodesulfurization process to be predicted. The work planned for the current quarter (May 1990 to July 1990) includes the following activities: (1) Construct a cloning vector that can be used in Rhodococcus rhodochrous IGTS8 from the small cryptic plasmid found in Rhodococcus rhodochrous ATCC 190607; (2) Develop techniques for the genetic analysis of IGTS8; (3) Continue biochemical experiments, particularly those that may allow the identification of desulfurization-related enzymes; (4) Continue experiments with coal to determine the kinetics of organic sulfur removal.

  3. Rhodococcus equi (Prescottella equi) vaccines; the future of vaccine development.

    PubMed

    Giles, C; Vanniasinkam, T; Ndi, S; Barton, M D

    2015-09-01

    For decades researchers have been targeting prevention of Rhodococcus equi (Rhodococcus hoagui/Prescottella equi) by vaccination and the horse breeding industry has supported the ongoing efforts by researchers to develop a safe and cost effective vaccine to prevent disease in foals. Traditional vaccines including live, killed and attenuated (physical and chemical) vaccines have proved to be ineffective and more modern molecular-based vaccines including the DNA plasmid, genetically attenuated and subunit vaccines have provided inadequate protection of foals. Newer, bacterial vector vaccines have recently shown promise for R. equi in the mouse model. This article describes the findings of key research in R. equi vaccine development and looks at alternative methods that may potentially be utilised.

  4. Quantitative aspects of fecal Rhodococcus (Corynebacterium) equi in foals.

    PubMed Central

    Takai, S; Ohkura, H; Watanabe, Y; Tsubaki, S

    1986-01-01

    Quantitative aspects of fecal Rhodococcus (Corynebacterium) equi in newborn foals for 12 weeks after birth were investigated on two horse breeding farms. R. equi was found in the feces of foals during week 1 of life. The greatest numbers of R. equi were present in the feces of foals during the first 8 weeks of their lives, which coincides with the age when foals are most liable to be exposed to R. equi. PMID:3700632

  5. Pangenome and Phylogenomic Analysis of the Pathogenic Actinobacterium Rhodococcus equi

    PubMed Central

    Anastasi, Elisa; MacArthur, Iain; Scortti, Mariela; Alvarez, Sonsiray; Giguère, Steeve; Vázquez-Boland, José A.

    2016-01-01

    We report a comparative study of 29 representative genomes of the animal pathogen Rhodococcus equi. The analyses showed that R. equi is genetically homogeneous and clonal, with a large core genome accounting for ≈80% of an isolates’ gene content. An open pangenome, even distribution of accessory genes among the isolates, and absence of significant core–genome recombination, indicated that gene gain/loss is a main driver of R. equi genome evolution. Traits previously predicted to be important in R. equi physiology, virulence and niche adaptation were part of the core genome. This included the lack of a phosphoenolpyruvate:carbohydrate transport system (PTS), unique among the rhodococci except for the closely related Rhodococcus defluvii, reflecting selective PTS gene loss in the R. equi–R. defluvii sublineage. Thought to be asaccharolytic, rbsCB and glcP non-PTS sugar permease homologues were identified in the core genome and, albeit inefficiently, R. equi utilized their putative substrates, ribose and (irregularly) glucose. There was no correlation between R. equi whole-genome phylogeny and host or geographical source, with evidence of global spread of genomovars. The distribution of host-associated virulence plasmid types was consistent with the exchange of the plasmids (and corresponding host shifts) across the R. equi population, and human infection being zoonotically acquired. Phylogenomic analyses demonstrated that R. equi occupies a central position in the Rhodococcus phylogeny, not supporting the recently proposed transfer of the species to a new genus. PMID:27638249

  6. Cold-tolerant alkane-degrading Rhodococcus species from Antarctica

    SciTech Connect

    Bej, A.K.; Saul, D.; Aislabie, J.

    2000-07-01

    Bioremediation is a possible mechanism for clean-up of hydrocarbon-contaminated soils in the Antarctic. Microbes indigenous to the Antarctic are required that degrade the hydrocarbon contaminants found in the soil, and that are able to survive and maintain activity under in situ conditions. Alkane-degrading bacteria previously isolated from oil-contaminated soil from around Scott Base, Antarctica, grew on a number of n-alkanes from hexane (C6) through to eicosane (C20) and the branched alkane pristane. Mineralization of {sup 14}C-dodecane was demonstrated with four strains. Representative isolates were identified as Rhodococcus species using 16S rDNA sequence analysis. Rhodococcus spp. strains 5/14 and 7/1 grew at -2 C but numbers of viable cells declined when incubated t 37 C. Both strains appear to have the major cold-shock gene cspA. Partial nucleotide sequence analyses of the PCR-amplified cspA open reading frame from Rhodococcus spp. strains 5/14 and 7/1 were approximately 60% identical to cspA from Escherichia coli.

  7. Brevibacterium linens pBL33 and Rhodococcus rhodochrous pRC1 cryptic plasmids replicate in Rhodococcus sp. R312 (formerly Brevibacterium sp. R312).

    PubMed

    Bigey, F; Grossiord, B; Chan Kuo Chion, C K; Arnaud, A; Galzy, P

    1995-02-27

    The replication of two cryptic plasmids from Brevibacterium linens ATCC 9174 (pBL33) and Rhodococcus rhodochrous ATCC 4276 (pRC1) was investigated in Rhodococcus sp. R312 (formerly Brevibacterium sp. R312). The recombinant plasmids pSP33 (pBL33 derivative) and pSPC1 (pRC1 derivative) were found to be suitable for establishing new host-vector systems for Rhodococcus sp. R312. They all carry the Tn903 neomycin-resistance-encoding gene (aphI).

  8. Pangenome and Phylogenomic Analysis of the Pathogenic Actinobacterium Rhodococcus equi.

    PubMed

    Anastasi, Elisa; MacArthur, Iain; Scortti, Mariela; Alvarez, Sonsiray; Giguère, Steeve; Vázquez-Boland, José A

    2016-10-23

    We report a comparative study of 29 representative genomes of the animal pathogen Rhodococcus equi The analyses showed that R. equi is genetically homogeneous and clonal, with a large core genome accounting for ≈80% of an isolates' gene content. An open pangenome, even distribution of accessory genes among the isolates, and absence of significant core-genome recombination, indicated that gene gain/loss is a main driver of R. equi genome evolution. Traits previously predicted to be important in R. equi physiology, virulence and niche adaptation were part of the core genome. This included the lack of a phosphoenolpyruvate:carbohydrate transport system (PTS), unique among the rhodococci except for the closely related Rhodococcus defluvii, reflecting selective PTS gene loss in the R. equi-R. defluvii sublineage. Thought to be asaccharolytic, rbsCB and glcP non-PTS sugar permease homologues were identified in the core genome and, albeit inefficiently, R. equi utilized their putative substrates, ribose and (irregularly) glucose. There was no correlation between R. equi whole-genome phylogeny and host or geographical source, with evidence of global spread of genomovars. The distribution of host-associated virulence plasmid types was consistent with the exchange of the plasmids (and corresponding host shifts) across the R. equi population, and human infection being zoonotically acquired. Phylogenomic analyses demonstrated that R. equi occupies a central position in the Rhodococcus phylogeny, not supporting the recently proposed transfer of the species to a new genus. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  9. Engineering of a xylose metabolic pathway in Rhodococcus strains.

    PubMed

    Xiong, Xiaochao; Wang, Xi; Chen, Shulin

    2012-08-01

    The two metabolically versatile actinobacteria Rhodococcus opacus PD630 and R. jostii RHA1 can efficiently convert diverse organic substrates into neutral lipids mainly consisting of triacylglycerol (TAG), the precursor of energy-rich hydrocarbon. Neither, however, is able to utilize xylose, the important component present in lignocellulosic biomass, as the carbon source for growth and lipid accumulation. In order to broaden their substrate utilization range, the metabolic pathway of d-xylose utilization was introduced into these two strains. This was accomplished by heterogenous expression of two well-selected genes, xylA, encoding xylose isomerase, and xylB, encoding xylulokinase from Streptomyces lividans TK23, under the control of the tac promoter with an Escherichia coli-Rhodococcus shuttle vector. The recombinant R. jostii RHA1 bearing xylA could grow on xylose as the sole carbon source, and additional expression of xylB further improved the biomass yield. The recombinant could consume both glucose and xylose in the sugar mixture, although xylose metabolism was still affected by the presence of glucose. The xylose metabolic pathway was also introduced into the high-lipid-producing strain R. opacus PD630 by expression of xylA and xylB. Under nitrogen-limited conditions, the fatty acid composition was determined, and lipid produced from xylose by recombinants of R. jostii RHA1 and R. opacus PD630 carrying xylA and xylB represented up to 52.5% and 68.3% of the cell dry weight (CDW), respectively. This work demonstrates that it is feasible to produce lipid from the sugars, including xylose, derived from renewable feedstock by genetic modification of rhodococcus strains.

  10. [Expression of acylamidase gene in Rhodococcus erythropolis strains].

    PubMed

    Lavrov, K V; Novikov, A D; Riabchenko, L E; Ianenko, A S

    2014-09-01

    The expression of a new acylamidase gene from R. erythropolis 37 was studied in Rhodococcus erythropolis strains. This acylamidase, as a result of its unique substrate specificity, can hydrolyse N-substituted amides (4'-nitroacetanilide, N-isopropylacrylamide, N'N-dimethylaminopropylacrylamide). A new expression system based on the use of the promoter region of nitrilhydratase genes from R. rhodochrous M8 was created to achieve constitutive synthesis of acylamidase in R. erythropolis cells. A fourfold improvement in the acylamidase activity of recombinant R. erythropolis cells as compared with the parent wild-type strain was obtained through the use of the new expression system.

  11. Conjugative transfer of cadmium resistance plasmids in Rhodococcus fascians strains.

    PubMed

    Desomer, J; Dhaese, P; Van Montagu, M

    1988-05-01

    The presence of a 138-kilobase plasmid (pD188) correlated with increased resistance to cadmium in Rhodococcus fascians D188. This plasmid could be transferred by a conjugation-like system in matings between R. fascians strains. Transconjugants expressed the cadmium resistance and could be used as donors in subsequent matings. Four other R. fascians strains (NCPPB 1488, NCPPB 1675, NCPPB 2551, and ATCC 12974) could also be used as donors for cadmium resistance in matings. Strain NCPPB 1675 showed a 100% cotransfer of cadmium and chloramphenicol resistance markers.

  12. pB264, a small, mobilizable, temperature sensitive plasmid from Rhodococcus

    PubMed Central

    Lessard, Philip A; O'Brien, Xian M; Currie, Devin H; Sinskey, Anthony J

    2004-01-01

    Background Gram-positive bacteria of the genus Rhodococcus have shown an extraordinary capacity for metabolizing recalcitrant organic compounds. One hindrance to the full exploitation of Rhodococcus is the dearth of genetic tools available for strain manipulation. To address this issue, we sought to develop a plasmid-based system for genetic manipulation of a variety of Rhodococcus strains. Results We isolated and sequenced pB264, a 4,970 bp cryptic plasmid from Rhodococcus sp. B264-1 with features of a theta-type replication mechanism. pB264 was nearly identical to pKA22, a previously sequenced but uncharacterized cryptic plasmid. Derivatives of pB264 replicate in a diverse range of Rhodococcus species, showing that this plasmid does not bear the same host range restrictions that have been exhibited by other theta replicating plasmids. Replication or maintenance of pB264 is inhibited at 37°C, making pB264 useful as a suicide vector for genetic manipulation of Rhodococcus. A series of deletions revealed that ca. 1.3 kb from pB264 was sufficient to support replication and stable inheritance of the plasmid. This region includes two open reading frames that encode functions (RepAB) that can support replication of pB264 derivatives in trans. Rhodococcus sp. B264-1 will mobilize pB264 into other Rhodococcus species via conjugation, making it possible to genetically modify bacterial strains that are otherwise difficult to transform. The cis-acting element (oriT) required for conjugal transfer of pB264 resides within a ca. 0.7 kb region that is distinct from the regions responsible for replication. Conclusion Shuttle vectors derived from pB264 will be useful for genetic studies and strain improvement in Rhodococcus, and will also be useful for studying the processes of theta replication and conjugal transfer among actinomycetes. PMID:15084226

  13. Rhodococcus sovatensis sp. nov., an actinomycete isolated from the hypersaline and heliothermal Lake Ursu.

    PubMed

    Táncsics, András; Máthé, István; Benedek, Tibor; Tóth, Erika M; Atasayar, Ewelina; Spröer, Cathrin; Márialigeti, Károly; Felföldi, Tamás; Kriszt, Balázs

    2017-02-01

    A Gram-stain-positive, strictly aerobic, mesophilic bacterium, designated H004T, was isolated from a water sample of the hypersaline and heliothermal Lake Ursu, Sovata, Romania. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain H004T formed a distinct phyletic lineage within the genus Rhodococcus. It shared the highest 16S rRNA gene sequence similarity with Rhodococcus yunnanensis YIM 70056T (98.80 %), followed by Rhodococcus fascians LMG 3623T (98.73 %), Rhodococcus cercidiphylli YIM 65003T (98.73 %), Rhodococcus cerastii C5T (98.58 %) and Rhodococcus kyotonensis DS472T (98.53 %). The alkB-based phylogenetic analysis further confirmed that this strain constitutes a highly unique lineage within the genus. Chemotaxonomic characteristics, including the predominant fatty acids acids C15 : 0, C18 : 1ω9c, C19 : 1ω11c/C19 : 1ω9c and C16 : 1ω7c/iso-C15 : 0 2-OH, the major quinone MK-8(H2), the presence of mycolic acids and cell-wall chemotype IV were also consistent with the properties of members of the genus Rhodococcus. The DNA G+C content of strain H004T was 65.4 mol%. The results of DNA-DNA hybridization analyses with the closest relatives, in combination with the alkB-based phylogenetic analysis, as well as the chemotaxonomic and physiological data, demonstrated that isolate H004T represents a novel species of the genus Rhodococcus, for which the name Rhodococcus sovatensissp. nov. is proposed. The type strain is H004T (=DSM 102881T=NCAIM B.02632T).

  14. Isolation and characterization of Rhodococcus sp. Y22 and its potential application to tobacco processing.

    PubMed

    Gong, Xiao-Wei; Yang, Jin-Kui; Duan, Yan-Qin; Dong, Jin-Yan; Zhe, Wei; Wang, Le; Li, Qing-Hua; Zhang, Ke-Qin

    2009-04-01

    A novel nicotine-degrading bacterium, strain Y22, was isolated and identified as Rhodococcus sp. Y22 based on its 16S rDNA sequence and morphological and biochemical features. The isolate could utilize nicotine as the sole source of carbon and nitrogen. Nicotine (1.0g/L) was degraded by Rhodococcus sp. Y22 within 52h at 28 degrees C and pH 7.0. Preparation of resting cells from nicotine-induced cultures was found to rapidly and efficiently degrade nicotine from solutions as well as from tobacco leaves. Therefore, Rhodococcus sp. Y22 has the potential to degrade nicotine during tobacco leave processing.

  15. Survival and replication of Rhodococcus equi in macrophages.

    PubMed Central

    Hondalus, M K; Mosser, D M

    1994-01-01

    Rhodococcus equi is a facultative intracellular bacterium of macrophages that can cause serious pneumonia in both young horses and immunocompromised people. Essential to understanding rhodococcus pathogenesis is a quantitative documentation of the intracellular events that follow macrophage phagocytosis of the organism. By using a bacterial immunofluorescence staining assay, we verified the intracellular survival and replicative potential of R. equi in both murine peritoneal macrophages and equine alveolar macrophages in vitro. Following an initial lag period of 6 to 12 h, the intracellular numbers of R. equi begin to rise, often reaching macrophage-compromising levels by 48 h. A quantitative determination of bacterial growth by a novel image analysis cytometry technique confirmed our fluorescence microscopic results. By 48 h postinfection, bacterial numbers had increased by more than fivefold, and the majority of infected macrophages in the monolayer contained 10 or more bacteria per cell. The intracellular organisms were viable, as evidenced by the ability to incorporate radiolabeled uracil. The use of these techniques has identified differences in the in vitro replicative capacities of a virulent strain and an avirulent strain of R. equi. A clinical isolate of R. equi expressing a 17-kDa virulence-associated plasmid-encoded antigen was able to survive and replicate within macrophages, whereas an avirulent, non-plasmid-containing strain replicated poorly. These results suggest that plasmid-encoded bacterial virulence factors may contribute to the ability of R. equi to replicate within its host cell, the macrophage. Images PMID:7927672

  16. Draft Genome of Rhodococcus rhodochrous TRN7, Isolated from the Coast of Trindade Island, Brazil

    PubMed Central

    Rodrigues, Edmo M.; Pylro, Victor S.; Dobbler, Priscila T.; Victoria, Filipe

    2016-01-01

    Here, we present a draft genome and annotation of Rhodococcus rhodochrous TRN7, isolated from Trindade Island, Brazil, which will provide genetic data to benefit the understanding of its metabolism. PMID:26941155

  17. Reclassification of Nocardia corynebacterioides Serrano et al. 1972 (Approved Lists 1980) as Rhodococcus corynebacterioides comb. nov.

    PubMed

    Yassin, A F; Schaal, K P

    2005-05-01

    The type strain of Nocardia corynebacterioides was the subject of a polyphasic taxonomic study. The 16S rRNA gene sequence was aligned with the sequences of representatives of the genera Corynebacterium, Dietzia, Gordonia, Mycobacterium, Nocardia, Rhodococcus, Skermania, Tsukamurella and Williamsia, and phylogenetic trees were constructed by using maximum-parsimony, maximum-likelihood and neighbour-joining methods. It was evident from the phylogenetic analysis that N. corynebacterioides represents a distinct phyletic line within the genus Rhodococcus. Menaquinone analysis showed that the organism contained dihydrogenated menaquinone with eight isoprene units, MK-8(H(2)), as the major isoprenologue. The genealogical evidence, together with chemotaxonomic and phenotypic data from this and previous studies, indicates that N. corynebacterioides DSM 20151(T) (= CIP 104510(T)) should be reclassified in the genus Rhodococcus as Rhodococcus corynebacterioides comb. nov.

  18. Draft genome sequence of Rhodococcus sp. strain P14, a biodegrader of high-molecular-weight polycyclic aromatic hydrocarbons.

    PubMed

    Zhang, Ying; Qin, Fujun; Qiao, Jing; Li, Gangmin; Shen, Chenghui; Huang, Tongwang; Hu, Zhong

    2012-07-01

    The genus Rhodococcus is known for its ability to degrade various xenobiotic compounds. Rhodococcus sp. strain P14 isolated from crude oil-contaminated sediments can degrade mineral oil and polycyclic aromatic hydrocarbons (PAHs). The draft genome sequence of Rhodococcus sp. P14 was obtained using Solexa technology, which provided an invaluable genetic background for further investigation of the ability of P14 to degrade xenobiotic compounds.

  19. Rhodococcus kroppenstedtii sp. nov., a novel actinobacterium isolated from a cold desert of the Himalayas, India.

    PubMed

    Mayilraj, S; Krishnamurthi, S; Saha, P; Saini, H S

    2006-05-01

    The taxonomic position of an actinomycete, strain K07-23T, isolated from a cold desert of the Himalayas, India, was established by a polyphasic approach. The strain exhibited phenotypic characters that were typical of the genus Rhodococcus. 16S rRNA gene sequence (1467 bases) comparisons confirmed that strain K07-23T belongs to the genus Rhodococcus. 16S rRNA sequence similarity studies showed that the isolate is very closely related to Nocardia corynebacterioides DSM 20151T (98.6 %), which has been recently reclassified as Rhodococcus corynebacterioides. It showed 94.4-96.6 % sequence similarity with other species of the genus Rhodococcus. However, genomic relatedness between strain K07-23T and R. corynebacterioides as revealed by DNA-DNA hybridization was low (62 %). Based on polyphasic analysis, strain K07-23T could be clearly distinguished from other species. It is proposed that strain K07-23T (=MTCC 6634T=DSM 44908T=JCM 13011T) represents a novel species of Rhodococcus, Rhodococcus kroppenstedtii sp. nov.

  20. Genome and Phenotype Microarray Analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7: Genetic Determinants and Metabolic Abilities with Environmental Relevance

    PubMed Central

    D’Ursi, Pasqualina; Milanesi, Luciano; Di Canito, Alessandra; Zampolli, Jessica; Collina, Elena; Decorosi, Francesca; Viti, Carlo; Fedi, Stefano; Presentato, Alessandro; Zannoni, Davide; Di Gennaro, Patrizia

    2015-01-01

    In this paper comparative genome and phenotype microarray analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7 were performed. Rhodococcus sp. BCP1 was selected for its ability to grow on short-chain n-alkanes and R. opacus R7 was isolated for its ability to grow on naphthalene and on o-xylene. Results of genome comparison, including BCP1, R7, along with other Rhodococcus reference strains, showed that at least 30% of the genome of each strain presented unique sequences and only 50% of the predicted proteome was shared. To associate genomic features with metabolic capabilities of BCP1 and R7 strains, hundreds of different growth conditions were tested through Phenotype Microarray, by using Biolog plates and plates manually prepared with additional xenobiotic compounds. Around one-third of the surveyed carbon sources was utilized by both strains although R7 generally showed higher metabolic activity values compared to BCP1. Moreover, R7 showed broader range of nitrogen and sulphur sources. Phenotype Microarray data were combined with genomic analysis to genetically support the metabolic features of the two strains. The genome analysis allowed to identify some gene clusters involved in the metabolism of the main tested xenobiotic compounds. Results show that R7 contains multiple genes for the degradation of a large set of aromatic and PAHs compounds, while a lower variability in terms of genes predicted to be involved in aromatic degradation was found in BCP1. This genetic feature can be related to the strong genetic pressure exerted by the two different environment from which the two strains were isolated. According to this, in the BCP1 genome the smo gene cluster involved in the short-chain n-alkanes degradation, is included in one of the unique regions and it is not conserved in the Rhodococcus strains compared in this work. Data obtained underline the great potential of these two Rhodococcus spp. strains for biodegradation and environmental decontamination

  1. Genome and Phenotype Microarray Analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7: Genetic Determinants and Metabolic Abilities with Environmental Relevance.

    PubMed

    Orro, Alessandro; Cappelletti, Martina; D'Ursi, Pasqualina; Milanesi, Luciano; Di Canito, Alessandra; Zampolli, Jessica; Collina, Elena; Decorosi, Francesca; Viti, Carlo; Fedi, Stefano; Presentato, Alessandro; Zannoni, Davide; Di Gennaro, Patrizia

    2015-01-01

    In this paper comparative genome and phenotype microarray analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7 were performed. Rhodococcus sp. BCP1 was selected for its ability to grow on short-chain n-alkanes and R. opacus R7 was isolated for its ability to grow on naphthalene and on o-xylene. Results of genome comparison, including BCP1, R7, along with other Rhodococcus reference strains, showed that at least 30% of the genome of each strain presented unique sequences and only 50% of the predicted proteome was shared. To associate genomic features with metabolic capabilities of BCP1 and R7 strains, hundreds of different growth conditions were tested through Phenotype Microarray, by using Biolog plates and plates manually prepared with additional xenobiotic compounds. Around one-third of the surveyed carbon sources was utilized by both strains although R7 generally showed higher metabolic activity values compared to BCP1. Moreover, R7 showed broader range of nitrogen and sulphur sources. Phenotype Microarray data were combined with genomic analysis to genetically support the metabolic features of the two strains. The genome analysis allowed to identify some gene clusters involved in the metabolism of the main tested xenobiotic compounds. Results show that R7 contains multiple genes for the degradation of a large set of aromatic and PAHs compounds, while a lower variability in terms of genes predicted to be involved in aromatic degradation was found in BCP1. This genetic feature can be related to the strong genetic pressure exerted by the two different environment from which the two strains were isolated. According to this, in the BCP1 genome the smo gene cluster involved in the short-chain n-alkanes degradation, is included in one of the unique regions and it is not conserved in the Rhodococcus strains compared in this work. Data obtained underline the great potential of these two Rhodococcus spp. strains for biodegradation and environmental decontamination

  2. An Adenoviral Vector Based Vaccine for Rhodococcus equi

    PubMed Central

    Giles, Carla; Ndi, Olasumbo; Barton, Mary D.; Vanniasinkam, Thiru

    2016-01-01

    Rhodococcus equi is a respiratory pathogen which primarily infects foals and is endemic on farms around the world with 50% mortality and 80% morbidity in affected foals. Unless detected early and treated appropriately the disease can be fatal. Currently, there is no vaccine available to prevent this disease. For decades researchers have endeavoured to develop an effective vaccine to no avail. In this study a novel human adenoviral vector vaccine for R. equi was developed and tested in the mouse model. This vaccine generated a strong antibody and cytokine response and clearance of R. equi was demonstrated following challenge. These results show that this vaccine could potentially be developed further for use as a vaccine to prevent R. equi disease in foals. PMID:27008624

  3. An Adenoviral Vector Based Vaccine for Rhodococcus equi.

    PubMed

    Giles, Carla; Ndi, Olasumbo; Barton, Mary D; Vanniasinkam, Thiru

    2016-01-01

    Rhodococcus equi is a respiratory pathogen which primarily infects foals and is endemic on farms around the world with 50% mortality and 80% morbidity in affected foals. Unless detected early and treated appropriately the disease can be fatal. Currently, there is no vaccine available to prevent this disease. For decades researchers have endeavoured to develop an effective vaccine to no avail. In this study a novel human adenoviral vector vaccine for R. equi was developed and tested in the mouse model. This vaccine generated a strong antibody and cytokine response and clearance of R. equi was demonstrated following challenge. These results show that this vaccine could potentially be developed further for use as a vaccine to prevent R. equi disease in foals.

  4. Biofilm development of the polyethylene-degrading bacterium Rhodococcus ruber.

    PubMed

    Sivan, A; Szanto, M; Pavlov, V

    2006-09-01

    We have recently isolated a biofilm-producing strain (C208) of Rhodococcus ruber that degraded polyethylene at a rate of 0.86% per week (r2=0.98). Strain C208 adheres to polyethylene immediately upon exposure to the polyolefin. This initial biofilm differentiates (in a stepwise process that lasts about 20 h) into cell-aggregation-forming microcolonies. Further organization yields "mushroom-like" three-dimensional structures on the mature biofilm. The ratio between the population densities of the biofilm and the planktonic C208 cells after 10 days of incubation was about 60:1, indicating a high preference for the biofilm mode of growth. Analysis of extracellular polymeric substances (EPS) in the biofilm of C208 revealed that the polysaccharides level was up to 2.5 folds higher than that of the protein. The biofilm showed a high viability even after 60 days of incubation, apparently due to polyethylene biodegradation.

  5. Conjugative transfer of cadmium resistance plasmids in Rhodococcus fascians strains

    SciTech Connect

    Desomer, J.; Dhaese, P.; Montagu, M.V.

    1988-05-01

    The presence of a 138-kilobase plasmid (pD188) correlated with increased resistance to cadmium in Rhodococcus fascians D188. This plasmid could be transferred by a conjugation-like system in matings between R. fascians strains. To examine this correlation we used large /sup 32/P-labeled pD188 subclones as probes in hybridization analyses with Southern blots of restricted total DNAs of D188 and its derivative mutants. Transconjugants expressed the cadmium resistance and could be used as donors in subsequent matings. Four other R. fascians strains (NCPPB 1488, NCPPB 1675, NCPPB 2551, and ATCC 12974) could also be used as donors for cadmium resistance in matings. Strain NCPPB 1675 showed a 100% cotransfer of cadmium and chloramphenicol resistance markers.

  6. Chromosomal locus that affects pathogenicity of Rhodococcus fascians.

    PubMed

    Vereecke, Danny; Cornelis, Karen; Temmerman, Wim; Jaziri, Mondher; Van Montagu, Marc; Holsters, Marcelle; Goethals, Koen

    2002-02-01

    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.

  7. Biotransformation of heterocyclic dinitriles by Rhodococcus erythropolis and fungal nitrilases.

    PubMed

    Vejvoda, Vojtech; Sveda, Ondrej; Kaplan, Ondrej; Prikrylová, Vera; Elisáková, Veronika; Himl, Michal; Kubác, David; Pelantová, Helena; Kuzma, Marek; Kren, Vladimír; Martínková, Ludmila

    2007-07-01

    2,6-Pyridinedicarbonitrile (1a) and 2,4-pyridinedicarbonitrile (2a) were hydrated by Rhodococcus erythropolis A4 to 6-cyanopyridine-2-carboxamide (1b; 83% yield) and 2-cyanopyridine-4-carboxamide (2b; 97% yield), respectively, after 10 min. After 118 h, the intermediates 1b or 2b were transformed into 2,6-pyridinedicarboxamide (1c; 35% yield) and 2,6-pyridinedicarboxylic acid (1d; 60% yield) or 2-cyanopyridine-4-carboxylic acid (2c; 64% yield), respectively. The nitrilase from Fusarium solani afforded cyanocarboxylic acids 1e and 2c after 118 h (yields 95 and 62%, respectively). 3,4-Pyridinedicarbonitrile (3a) and 2,3-pyrazinedicarbonitrile (4a) were inferior substrates of nitrile hydratase and nitrilase.

  8. Initial Transformations in the Biodegradation of Benzothiazoles by Rhodococcus Isolates

    PubMed Central

    De Wever, Helene; Vereecken, Karen; Stolz, Andreas; Verachtert, Hubert

    1998-01-01

    Benzothiazole-2-sulfonate (BTSO3) is one of the side products occurring in 2-mercaptobenzothiazole (MBT) production wastewater. We are the first to isolate an axenic culture capable of BTSO3 degradation. The isolate was identified as a Rhodococcus erythropolis strain and also degraded 2-hydroxybenzothiazole (OBT) and benzothiazole (BT), but not MBT, which was found to inhibit the biodegradation of OBT, BT, and BTSO3. In anaerobic resting cell assays, BTSO3 was transformed into OBT in stoichiometric amounts. Under aerobic conditions, OBT was observed as an intermediate in BT breakdown and an unknown compound transiently accumulated in several assays. This product was identified as a dihydroxybenzothiazole. Benzothiazole degradation pathways seem to converge into OBT, which is then transformed further into the dihydroxy derivative. PMID:9726870

  9. Degradation of a mixture of hydrocarbons, gasoline, and diesel oil additives by Rhodococcus aetherivorans and Rhodococcus wratislaviensis.

    PubMed

    Auffret, Marc; Labbé, Diane; Thouand, Gérald; Greer, Charles W; Fayolle-Guichard, Françoise

    2009-12-01

    Two strains, identified as Rhodococcus wratislaviensis IFP 2016 and Rhodococcus aetherivorans IFP 2017, were isolated from a microbial consortium that degraded 15 petroleum compounds or additives when provided in a mixture containing 16 compounds (benzene, toluene, ethylbenzene, m-xylene, p-xylene, o-xylene, octane, hexadecane, 2,2,4-trimethylpentane [isooctane], cyclohexane, cyclohexanol, naphthalene, methyl tert-butyl ether [MTBE], ethyl tert-butyl ether [ETBE], tert-butyl alcohol [TBA], and 2-ethylhexyl nitrate [2-EHN]). The strains had broad degradation capacities toward the compounds, including the more recalcitrant ones, MTBE, ETBE, isooctane, cyclohexane, and 2-EHN. R. wratislaviensis IFP 2016 degraded and mineralized to different extents 11 of the compounds when provided individually, sometimes requiring 2,2,4,4,6,8,8-heptamethylnonane (HMN) as a cosolvent. R. aetherivorans IFP 2017 degraded a reduced spectrum of substrates. The coculture of the two strains degraded completely 13 compounds, isooctane and 2-EHN were partially degraded (30% and 73%, respectively), and only TBA was not degraded. Significant MTBE and ETBE degradation rates, 14.3 and 116.1 mumol of ether degraded h(-1) g(-1) (dry weight), respectively, were measured for R. aetherivorans IFP 2017. The presence of benzene, toluene, ethylbenzene, and xylenes (BTEXs) had a detrimental effect on ETBE and MTBE biodegradation, whereas octane had a positive effect on the MTBE biodegradation by R. wratislaviensis IFP 2016. BTEXs had either beneficial or detrimental effects on their own degradation by R. wratislaviensis IFP 2016. Potential genes involved in hydrocarbon degradation in the two strains were identified and partially sequenced.

  10. Degradation of a Mixture of Hydrocarbons, Gasoline, and Diesel Oil Additives by Rhodococcus aetherivorans and Rhodococcus wratislaviensis▿

    PubMed Central

    Auffret, Marc; Labbé, Diane; Thouand, Gérald; Greer, Charles W.; Fayolle-Guichard, Françoise

    2009-01-01

    Two strains, identified as Rhodococcus wratislaviensis IFP 2016 and Rhodococcus aetherivorans IFP 2017, were isolated from a microbial consortium that degraded 15 petroleum compounds or additives when provided in a mixture containing 16 compounds (benzene, toluene, ethylbenzene, m-xylene, p-xylene, o-xylene, octane, hexadecane, 2,2,4-trimethylpentane [isooctane], cyclohexane, cyclohexanol, naphthalene, methyl tert-butyl ether [MTBE], ethyl tert-butyl ether [ETBE], tert-butyl alcohol [TBA], and 2-ethylhexyl nitrate [2-EHN]). The strains had broad degradation capacities toward the compounds, including the more recalcitrant ones, MTBE, ETBE, isooctane, cyclohexane, and 2-EHN. R. wratislaviensis IFP 2016 degraded and mineralized to different extents 11 of the compounds when provided individually, sometimes requiring 2,2,4,4,6,8,8-heptamethylnonane (HMN) as a cosolvent. R. aetherivorans IFP 2017 degraded a reduced spectrum of substrates. The coculture of the two strains degraded completely 13 compounds, isooctane and 2-EHN were partially degraded (30% and 73%, respectively), and only TBA was not degraded. Significant MTBE and ETBE degradation rates, 14.3 and 116.1 μmol of ether degraded h−1 g−1 (dry weight), respectively, were measured for R. aetherivorans IFP 2017. The presence of benzene, toluene, ethylbenzene, and xylenes (BTEXs) had a detrimental effect on ETBE and MTBE biodegradation, whereas octane had a positive effect on the MTBE biodegradation by R. wratislaviensis IFP 2016. BTEXs had either beneficial or detrimental effects on their own degradation by R. wratislaviensis IFP 2016. Potential genes involved in hydrocarbon degradation in the two strains were identified and partially sequenced. PMID:19837842

  11. Rhodococcus yunnanensis sp. nov., a mesophilic actinobacterium isolated from forest soil.

    PubMed

    Zhang, Yu-Qin; Li, Wen-Jun; Kroppenstedt, Reiner M; Kim, Chang-Jin; Chen, Guo-Zhong; Park, Dong-Jin; Xu, Li-Hua; Jiang, Cheng-Lin

    2005-05-01

    A Gram-positive, aerobic, non-motile, mesophilic strain, designated YIM 70056(T), was isolated from a forest soil sample in Yunnan Province, China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that this isolate had less than 97.0 % similarity to any Rhodococcus species with validly published names, with the exception of Rhodococcus fascians (DSM 20669(T)), which was found to be its closest neighbour (98.9 % similarity). Chemotaxonomic data, including peptidoglycan type, diagnostic sugar compositions, fatty acid profiles, menaquinones, polar lipids and mycolic acids, were determined for this isolate; the results supported the affiliation of strain YIM 70056(T) to the genus Rhodococcus. The DNA G + C content was 63.5 mol%. The results of DNA-DNA hybridization with R. fascians DSM 20669(T), in combination with chemotaxonomic and physiological data, demonstrated that isolate YIM 70056(T) represents a novel Rhodococcus species, for which the name Rhodococcus yunnanensis sp. nov. is proposed, with YIM 70056(T) (=CCTCC AA 204007(T) = KCTC 19021(T) = DSM 44837(T)) as the type strain.

  12. Rhodococcus cercidiphylli sp. nov., a new endophytic actinobacterium isolated from a Cercidiphyllum japonicum leaf.

    PubMed

    Li, Jie; Zhao, Guo-Zhen; Chen, Hua-Hong; Qin, Sheng; Xu, Li-Hua; Jiang, Cheng-Lin; Li, Wen-Jun

    2008-06-01

    An endophytic actinobacterium, designated YIM 65003(T), was isolated from a surface sterilized leaf sample of Cercidiphyllum japonicum collected from Yunnan province, south-west China. The morphological and chemotaxonomic properties of the isolate were typical of members of the genus Rhodococcus. Analysis of the 16S rRNA gene sequence revealed that the isolate was most closely related to Rhodococcus fascians DSM 20669(T) (99.6%) and Rhodococcus yunnanensis YIM 70056(T) (99.0%). DNA-DNA hybridization with the above microorganisms (46.3% and 48.8%, respectively), in combination with differences in the biochemical and physiological properties, suggested that strain YIM 65003(T) should be classified within a novel species of the genus Rhodococcus, for which the name Rhodococcus cercidiphylli sp. nov. is proposed, with YIM 65003(T) (=CCTCC AB 207160(T)=DSM 45141(T)) as the type strain. The 16S rRNA gene sequence of strain YIM 65003(T) has been deposited in GenBank under the accession number EU325542.

  13. Biodesulphurization of gasoline by Rhodococcus erythropolis supported on polyvinyl alcohol.

    PubMed

    Fatahi, A; Sadeghi, S

    2017-05-01

    A new biodesulphurization (BDS) method has been considered using Rhodococcus erythropolis supported on polyvinyl alcohol (PVA) for BDS of thiophene as a gasoline sulphur model compound in n-hexane as the solvent, subsequently this biocatalyst has been applied to BDS of gasoline samples. The obtained results according to UV-Spectrophotometer analysis at 240 nm showed that 97·41% of thiophene at the optimum condition of primary concentration 80 mg l(-1) , pH = 7, by 0·1 g of biocatalyst in 30°C and after 20 h of contact time has been degraded. These optimum conditions have been applied to gasoline BDS and the biodegradation of gasoline thiophenic compounds have been investigated by gas chromatography-mass spectrometry (GC-MS). According to GC-MS, thiophene and its 2-methyl, 3-methyl and 2- ethyl derivatives had acceptable biodegradation efficiencies of about 26·67, 21·03, 23·62% respectively. Also, benzothiophene that has been detected in a gasoline sample had 38·89% biodegradation efficiency at optimum conditions, so biomodification of PVA by R. erythropolis produces biocatalysts with an active metabolism that facilitates the interaction of bacterial strain with gasoline thiophenic compounds. The morphology and surface functional groups of supported R. erythropolis on PVA have been investigated by scanning electron microscope (SEM) and FT-IR spectroscopy respectively. SEM images suggest some regular layered shape for the supported bacteria. FT-IR spectra indicate a desirable interaction between bacterial cells and polymer supports. Also, the recovery of biocatalyst has been investigated and after three times of using in BDS activity, its biocatalytic ability had no significant decreases. The biomodification of polyvinyl alcohol by Rhodococcus erythropolis described herein produces a new biocatalyst which can be used for significantly reducing the thiophenic compounds of gasoline and other fossil fuels. The immobilization process is to increase the

  14. The correct name of the taxon that contains the type strain of Rhodococcus equi.

    PubMed

    Tindall, B J

    2014-01-01

    Based on a nomenclatural point of view, the name Rhodococcus equi is associated, as required by the Bacteriological Code, with a defined position, rank and circumscription. A search of the literature indicates that the name Rhodococcus equi (Magnusson 1923) Goodfellow and Alderson 1977 has also been shown to be a synonym of Corynebacterium equi Magnusson 1923, Corynebacterium hoagii (Morse 1912) Eberson 1918 and Nocardia restricta (Turfitt 1944) McClung 1974. Application of the rules of the Bacteriological Code together with the currently inferred taxonomic concept associated with the species bearing the name Rhodococcus equi indicates that this is not the correct name of this taxon and the use of that name in the context of a circumscription that includes the type strain of the species Corynebacterium hoagii is contrary to the Rules of the Code.

  15. Sulfur-selective desulfurization of dibenzothiophene and diesel oil by newly isolated Rhodococcus sp. strains.

    PubMed

    Castorena, Gladys; Suárez, Claudia; Valdez, Idania; Amador, Guadalupe; Fernández, Luis; Le Borgne, Sylvie

    2002-09-24

    New desulfurizing bacteria able to convert dibenzothiophene into 2-hydroxybiphenyl and sulfate were isolated from contaminated soils collected in Mexican refineries. Random amplified polymorphic DNA analysis showed they were different from previously reported Rhodococcus erythropolis desulfurizing strains. According to 16S rRNA gene sequencing and fatty acid analyses, these new isolates belonged to the genus Rhodococcus. These strains could desulfurize 4,6-dimethyldibenzothiophene which is one of the most difficult dibenzothiophene derivatives to remove by hydrodesulfurization. A deeply hydrodesulfurized diesel oil containing significant amounts of 4,6-dimethyldibenzothiophene was treated with Rhodococcus sp. IMP-S02 cells. Up to 60% of the total sulfur was removed and all the 4,6-dimethyldibenzothiophene disappeared as a result of this treatment.

  16. A 2-Hydroxypyridine Catabolism Pathway in Rhodococcus rhodochrous Strain PY11

    PubMed Central

    Gasparavičiūtė, Renata; Rutkienė, Rasa; Tauraitė, Daiva; Meškys, Rolandas

    2015-01-01

    Rhodococcus rhodochrous PY11 (DSM 101666) is able to use 2-hydroxypyridine as a sole source of carbon and energy. By investigating a gene cluster (hpo) from this bacterium, we were able to reconstruct the catabolic pathway of 2-hydroxypyridine degradation. Here, we report that in Rhodococcus rhodochrous PY11, the initial hydroxylation of 2-hydroxypyridine is catalyzed by a four-component dioxygenase (HpoBCDF). A product of the dioxygenase reaction (3,6-dihydroxy-1,2,3,6-tetrahydropyridin-2-one) is further oxidized by HpoE to 2,3,6-trihydroxypyridine, which spontaneously forms a blue pigment. In addition, we show that the subsequent 2,3,6-trihydroxypyridine ring opening is catalyzed by the hypothetical cyclase HpoH. The final products of 2-hydroxypyridine degradation in Rhodococcus rhodochrous PY11 are ammonium ion and α-ketoglutarate. PMID:26655765

  17. A 2-Hydroxypyridine Catabolism Pathway in Rhodococcus rhodochrous Strain PY11.

    PubMed

    Vaitekūnas, Justas; Gasparavičiūtė, Renata; Rutkienė, Rasa; Tauraitė, Daiva; Meškys, Rolandas

    2015-12-11

    Rhodococcus rhodochrous PY11 (DSM 101666) is able to use 2-hydroxypyridine as a sole source of carbon and energy. By investigating a gene cluster (hpo) from this bacterium, we were able to reconstruct the catabolic pathway of 2-hydroxypyridine degradation. Here, we report that in Rhodococcus rhodochrous PY11, the initial hydroxylation of 2-hydroxypyridine is catalyzed by a four-component dioxygenase (HpoBCDF). A product of the dioxygenase reaction (3,6-dihydroxy-1,2,3,6-tetrahydropyridin-2-one) is further oxidized by HpoE to 2,3,6-trihydroxypyridine, which spontaneously forms a blue pigment. In addition, we show that the subsequent 2,3,6-trihydroxypyridine ring opening is catalyzed by the hypothetical cyclase HpoH. The final products of 2-hydroxypyridine degradation in Rhodococcus rhodochrous PY11 are ammonium ion and α-ketoglutarate.

  18. Microbial desulfurization of gasoline by free whole-cells of Rhodococcus erythropolis XP.

    PubMed

    Yu, Bo; Ma, Cuiqing; Zhou, Wenjuan; Wang, Ying; Cai, Xiaofeng; Tao, Fei; Zhang, Quan; Tong, Mingyou; Qu, Jingyao; Xu, Ping

    2006-05-01

    Rhodococcus erythropolis XP could grow well with condensed thiophenes, mono-thiophenic compounds and mercaptans present in gasoline. Rhodococcus erythropolis XP was also capable of efficiently degrading the condensed thiophenes in resting cell as well as biphasic reactions in which n-octane served as a model oil phase. Free whole cells of R. erythropolis XP were adopted to desulfurize fluid catalytic cracking (FCC) and straight-run (SR) gasoline oils. About 30% of the sulfur content of FCC gasoline and 85% of sulfur in SR gasoline were reduced, respectively. Gas chromatography analysis with atomic emission detection also showed depletion of sulfur compounds in SR gasoline. Rhodococcus erythropolis XP could partly resist the toxicity of gasoline and had an application potential to biodesulfurization of gasoline.

  19. [Antiadhesive potencial of Rhodococcus erythropolis IMB Ac-5017 biosurfactants].

    PubMed

    Pirog, T P; Gritsenko, N A; Konon, A D; Shevchuk, T A; Iutinskaia, G A

    2014-01-01

    The effect of Rhodococcus erythropolis IMB Ac-5017 biosurfactants (surface-active substances, SAS) with different degree of purification on attachment of bacteria (Escherichia coli IEM-1, Bacillus subtilis BT-2, Proteus vulgaris BT-1, Staphylococcus aureus BMC-1, Pseudomonas aeruginosa P-55, Enterobacter cloacae AC-22, Erwinia aroidaeae B-433), yeasts (Candida albicans D-6) and fungi (Aspergillus niger P-3, Fusarium culmorum T-7) to the abiotic surfaces (glass, plastic, ceramics, steel, linoleum) was studied. The dependence of microorganisms adhesion on degree of SAS purification (supernatant, purified SAS solution), SAS concentration (0,04-1,25 mg/ml), type of surface and test-cultures was established. The adhesion of majority investigated bacterial cells after treatment of abiotic surfaces with supernatant of cultural liquid with SAS concentration 0,06-0,25 mg/ml was on the average 20-45, yeasts C. albicans D-6--30-75% and was less than that purified SAS solution with the same concentration. Higher antiadhesive activity of supernatant as compared to purified SAS solution testifies to possibility of exception of the expensive stage of isolation and purification at obtaining of preparations with antiadhesive properties.

  20. Molecular characterization of Rhodococcus equi isolates in equines

    PubMed Central

    Javed, Rabyia; Taku, A. K.; Sharma, R. K.; Badroo, Gulzaar Ahmed

    2017-01-01

    Aim: The aim was to determine the occurrence of Rhodococcus equi in equines and their environment in Jammu (R.S. Pura, Katra), molecular characterization and to determine the antibiotic resistance pattern of R. equi. Materials and Methods: A total of 96 nasopharyngeal swab samples were collected from equines. The organism was isolated on Columbia nalidixic acid agar containing 5% sheep blood as well as on sheep blood agar and was later confirmed by cultural characteristics and biochemical tests. Molecular detection of R. equi isolates was done by 16S rRNA gene amplification followed by virulence associated protein A (Vap A) gene amplification. Antibiogram was performed against five antibiotics, viz., amoxicillin, penicillin G, streptomycin, rifampicin, and methicillin. Results: During the study, 9 R. equi isolates were identified on the basis of cultural and biochemical tests. In the polymerase chain reaction based detection, 3 among the 9 rhodococcal isolates were positive for species-specific 16S rRNA gene and revealed amplicon of 450 bp for confirmation of 16S rRNA gene. None of the sample was found positive for Vap A gene. In antibiogram, R. equi isolates were found sensitive for amoxicillin, while some isolates were also found resistant to the most conventional antibiotic penicillin G. Conclusion: From this study, it was concluded that R. equi infection is prevalent in equines in Jammu region of India and the indiscriminate use of the antibiotics is leading toward the development of resistant strains of R. equi. PMID:28246441

  1. Activity of 10 antimicrobial agents against intracellular Rhodococcus equi.

    PubMed

    Giguère, Steeve; Berghaus, Londa J; Lee, Elise A

    2015-08-05

    Studies with facultative intracellular bacterial pathogens have shown that evaluation of the bactericidal activity of antimicrobial agents against intracellular bacteria is more closely associated with in vivo efficacy than traditional in vitro susceptibility testing. The objective of this study was to determine the relative activity of 10 antimicrobial agents against intracellular Rhodococcus equi. Equine monocyte-derived macrophages were infected with virulent R. equi and exposed to erythromycin, clarithromycin, azithromycin, rifampin, ceftiofur, gentamicin, enrofloxacin, vancomycin, imipenem, or doxycycline at concentrations achievable in plasma at clinically recommended dosages in foals. The number of intracellular R. equi was determined 48h after infection by counting colony forming units (CFUs). The number of R. equi CFUs in untreated control wells were significantly higher than those of monolayers treated with antimicrobial agents. Numbers of R. equi were significantly lower in monolayers treated with enrofloxacin followed by those treated with gentamicin, and vancomycin, when compared to monolayers treated with other antimicrobial agents. Numbers of R. equi in monolayers treated with doxycycline were significantly higher than those of monolayers treated with other antimicrobial agents. Differences in R. equi CFUs between monolayers treated with other antimicrobial agents were not statistically significant. Enrofloxacin, gentamicin, and vancomycin are the most active drugs in equine monocyte-derived macrophages infected with R. equi. Additional studies will be needed to determine if these findings correlate with in vivo efficacy. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Molecular characterization of Rhodococcus equi isolates in equines.

    PubMed

    Javed, Rabyia; Taku, A K; Sharma, R K; Badroo, Gulzaar Ahmed

    2017-01-01

    The aim was to determine the occurrence of Rhodococcus equi in equines and their environment in Jammu (R.S. Pura, Katra), molecular characterization and to determine the antibiotic resistance pattern of R. equi. A total of 96 nasopharyngeal swab samples were collected from equines. The organism was isolated on Columbia nalidixic acid agar containing 5% sheep blood as well as on sheep blood agar and was later confirmed by cultural characteristics and biochemical tests. Molecular detection of R. equi isolates was done by 16S rRNA gene amplification followed by virulence associated protein A (Vap A) gene amplification. Antibiogram was performed against five antibiotics, viz., amoxicillin, penicillin G, streptomycin, rifampicin, and methicillin. During the study, 9 R. equi isolates were identified on the basis of cultural and biochemical tests. In the polymerase chain reaction based detection, 3 among the 9 rhodococcal isolates were positive for species-specific 16S rRNA gene and revealed amplicon of 450 bp for confirmation of 16S rRNA gene. None of the sample was found positive for Vap A gene. In antibiogram, R. equi isolates were found sensitive for amoxicillin, while some isolates were also found resistant to the most conventional antibiotic penicillin G. From this study, it was concluded that R. equi infection is prevalent in equines in Jammu region of India and the indiscriminate use of the antibiotics is leading toward the development of resistant strains of R. equi.

  3. Phenotypic and genotypic characterization of Rhodococcus equi isolated from sputum.

    PubMed

    Silva, Paulo da; Santos, Adolfo Carlos Barreto; Sato, Daisy Nakamura; Silva, Jaqueline Otero; Medeiros, Marta Inês Cazentini; Carneiro, Ana Maria Machado; Leite, Sergio Roberto de Andrade; Leite, Clarice Queico Fujimura

    2012-01-01

    Rhodococcus equi is an opportunistic pathogen, causing rhodococcosis, a condition that can be confused with tuberculosis. Often, without identifying M. tuberculosis, physicians initiate empiric treatment for tuberculosis. R. equi and M. tuberculosis have different susceptibility to drugs. Identification of R. equi is based on a variety of phenotypic, chromatographic, and genotypic characteristics. This study aimed to characterize bacterial isolates from sputum samples suggestive of R. equi. The phenotypic identification included biochemical assays; thin-layer chromatography (TLC) and polymerase chain reaction (PCR) were used for genotypic identification. Among 78 Gram-positive and partially acid-fast bacilli isolated from the sputum of tuberculosis-suspected patients, 51 were phenotypically and genotypically characterized as R. equi based on literature data. Mycolic acid analysis showed that all suspected R. equi had compounds with a retention factor (R(f)) between 0.4-0.5. Genotypic characterization indicated the presence of the choE gene 959bp fragments in 51 isolates CAMP test positive. Twenty-two CAMP test negative isolates were negative for the choE gene. Five isolates presumptively identified as R. equi, CAMP test positive, were choE gene negative, and probably belonged to other bacterial species. The phenotypic and molecular techniques used constitute a good methodological tool to identify R. equi. Copyright © 2012 Elsevier Editora Ltda. All rights reserved.

  4. Rhodococcus equi: the many facets of a pathogenic actinomycete.

    PubMed

    Vázquez-Boland, José A; Giguère, Steeve; Hapeshi, Alexia; MacArthur, Iain; Anastasi, Elisa; Valero-Rello, Ana

    2013-11-29

    Rhodococcus equi is a soil-dwelling pathogenic actinomycete that causes pulmonary and extrapulmonary pyogranulomatous infections in a variety of animal species and people. Young foals are particularly susceptible and develop a life-threatening pneumonic disease that is endemic at many horse-breeding farms worldwide. R. equi is a facultative intracellular parasite of macrophages that replicates within a modified phagocytic vacuole. Its pathogenicity depends on a virulence plasmid that promotes intracellular survival by preventing phagosome-lysosome fusion. Species-specific tropism of R. equi for horses, pigs and cattle appears to be determined by host-adapted virulence plasmid types. Molecular epidemiological studies of these plasmids suggest that human R. equi infection is zoonotic. Analysis of the recently determined R. equi genome sequence has identified additional virulence determinants on the bacterial chromosome. This review summarizes our current understanding of the clinical aspects, biology, pathogenesis and immunity of this fascinating microbe with plasmid-governed infectivity. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Disseminated Rhodococcus equi infection in dromedary camels (Camelus dromedarius).

    PubMed

    Kinne, J; Madarame, H; Takai, S; Jose, S; Wernery, U

    2011-04-21

    Rhodococcus (R). equi, a recognized pathogen in horses, is emerging as a human opportunistic pathogen, especially in immunocompromized people. It affects also New World camelids, but there are no reports of R. equi infection in Old World camelids yet. Four cases of disseminated R. equi infection in adult breeding dromedaries occurred at one camel farm near Dubai within 16 months of each other. At necropsy the lungs were diffusely consolidated with large caseous areas. Histology revealed severe suppurative to necrotising pneumonia with multiple encapsulated abscesses. Immunohistochemistry enabled the detection of 15- to 17-kDa antigens (VapA) of R. equi in the lung sections. High numbers of R. equi were isolated from the lung lesions as well as from liver, spleen and mediastinal lymph nodes, indicative of septicaemia. The isolated strains were PCR-positive for the specific virulence plasmid (VapA-Gen) of R. equi, indicating virulent strains and containing an 85-kb type I plasmid. This is the first report of disseminated R. equi infection in Old World camelids. Since adult camels in general do not suffer from bacterial caused pneumonia (except tuberculosis), this is a new emerging disease for camels. Copyright © 2010 Elsevier B.V. All rights reserved.

  6. Radiologic features of Rhodococcus equi pneumonia in AIDS.

    PubMed

    Muntaner, L; Leyes, M; Payeras, A; Herrera, M; Gutierrez, A

    1997-01-01

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

  7. The leafy gall syndrome induced by Rhodococcus fascians.

    PubMed

    Stes, Elisabeth; Francis, Isolde; Pertry, Ine; Dolzblasz, Alicja; Depuydt, Stephen; Vereecke, Danny

    2013-05-01

    The Actinomycete Rhodococcus fascians causes the leafy gall syndrome, an infectious plant disease that affects a wide range of plants, primarily dicotyledonous herbs. The syndrome is associated with delayed senescence, loss of apical dominance, activation of dormant axillary meristems, and formation of multiple inflorescences, leading to a stunted and bushy plant appearance. A major breakthrough in the elucidation of the virulence strategy of this pathogen was the discovery of a linear virulence plasmid, pFiD188 for R. fascians strain D188. Upon perception of a compatible host plant, an autoregulatory mechanism mediated by the att operon directs a switch in the bacterial life style from a harmless epiphyte into a pathogenic endophyte and, concomitantly, activates gene expression of the fas operon that encodes a cytokinin biosynthesis pathway. A mixture of five cytokinins determines the cytokinin activity of R. fascians that directly affects plant responses and development. Moreover, the bacterial cytokinins stimulate the host to produce auxins and polyamines, that function as accessory signals to aid in symptom development. The plant reacts against the developmental hijacking by R. fascians by activating a set of counteracting measures that ultimately results in a delicate balance, allowing a long-lasting biotrophic interaction.

  8. Purification and characterization of limonoate dehydrogenase from Rhodococcus fascians.

    PubMed

    Humanes, L; López-Ruiz, A; Merino, M T; Roldán, J M; Diez, J

    1997-09-01

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

  9. Purification and characterization of limonoate dehydrogenase from Rhodococcus fascians.

    PubMed Central

    Humanes, L; López-Ruiz, A; Merino, M T; Roldán, J M; Diez, J

    1997-01-01

    Limonoate dehydrogenase from Rhodococcus fascians has been purified to electrophoretic homogeneity by a procedure that consists of ion-exchange, hydrophobic, and affinity chromatography. The native enzyme has a molecular mass of around 128,000 Da and appears to be composed of four similar subunits (30,000 Da each). The isoelectric point is 4.9 as determined by isoelectric focusing. The homogeneous enzyme was used to determine the NH2-terminal amino acid sequence. The enzyme was purified from cells grown in either fructose or limonoate as a carbon source. Limonoate dehydrogenase activity was higher in limonoate-grown cultures. Additionally, the enzyme preparations differed in their affinity for limonoids but not for NAD+. In all cases limonoate dehydrogenase exhibited a higher catalytic rate and stronger affinity for limonoate A-ring lactone than for disodium limonoate, the limonoid traditionally used for in vitro activity assays. Our data confirm previous reports proposing that limonoate A-ring lactone is the physiological substrate for limonoate dehydrogenase. The increase in limonoate dehydrogenase activity observed in limonoate-grown cultures appears to be caused by a rise in protein levels, since chloramphenicol prevented such an effect. PMID:9292989

  10. Kinetic mechanism of putrescine oxidase from Rhodococcus erythropolis.

    PubMed

    Kopacz, Malgorzata M; Heuts, Dominic P H M; Fraaije, Marco W

    2014-10-01

    Putrescine oxidase from Rhodococcus erythropolis (PuO) is a flavin-containing amine oxidase from the monoamine oxidase family that performs oxidative deamination of aliphatic diamines. In this study we report pre-steady-state kinetic analyses of the enzyme with the use of single- and double-mixing stopped-flow spectroscopy and putrescine as a substrate. During the fast and irreversible reductive half-reaction no radical intermediates were observed, suggesting a direct hydride transfer from the substrate to the FAD. The rate constant of flavin reoxidation depends on the ligand binding; when the imine product was bound to the enzyme the rate constant was higher than with free enzyme species. Similar results were obtained with product-mimicking ligands and this indicates that a ternary complex is formed during catalysis. The obtained kinetic data were used together with steady-state rate equations derived for ping-pong, ordered sequential and bifurcated mechanisms to explore which mechanism is operative. The integrated analysis revealed that PuO employs a bifurcated mechanism due to comparable rate constants of product release from the reduced enzyme and reoxidation of the reduced enzyme-product complex.

  11. Identification of indigo-related pigments produced by Escherichia coli containing a cloned Rhodococcus gene.

    PubMed

    Hart, S; Koch, K R; Woods, D R

    1992-01-01

    Pigments produced by Escherichia coli containing a cloned piece of DNA from Rhodococcus sp. ATCC 21145 were extracted in chloroform and separated into blue and pink components. Evidence from TLC, NMR spectroscopy, absorption spectrum analysis and solubility behaviour suggested that the blue pigment was indigo and the pink pigment was indirubin, a structural isomer of indigo. The proposed pathway for pigment production on LB agar involves the conversion of tryptophan to indole by tryptophanase of E. coli and the oxidation of indole to indigo by the product of the cloned Rhodococcus DNA insert.

  12. Disseminated Rhodococcus equi infection in a patient with Hodgkin lymphoma.

    PubMed

    Mikić, Dragan; Djordjević, Zoran; Sekulović, Leposava; Kojić, Miroslav; Tomanović, Branka

    2014-03-01

    Rhodococcus (R) equi is an opportunistic, uncommon human pathogen that causes mainly infection in immunocompromised hosts. The disease is usually presented as subacute pneumonia that is mostly cavitary and sometimes bacteremic. We reported the extremly rare case of a 43-year-old woman with Hodgkin lymphoma, who developed R equi pulmonary infection after recieving multiple courses of chemotherapy. Secondary, the patient developed bacteremia, leading to sepsis and dissemination of R equi infection in many extrapulmonary sites. At addmission the patient was febrile, tachypnoic, tachycardic, hypotensive, with fa cial edema, splenomegaly, positive meningeal signs, left hemiparesis and paraparesis. Laboratory data included erythrocyte sedimentation rate (ESR) > 140 mm/h, C-reactive protein (CRP) 143.0 mg/L, red blood cells (RBC) 2.14 x 10(12)/L, whyite blood cells (WBC) 2.8 x 10(9)/L, lactate dehydrogenase (LDH) 706 U/L, serum albumin 26 g/L, sodium 127 mmol/L and potassium 2.7 mmol/L. Blood culture and culture of sputum and empyema were positive for R equi. Imaging studies demonstrated a large right cavitary pneumonia and abscess, empyema, pericarditis, mediastinal and intra-abdominal lymphadenopathy, brain and psoas abscesses, osteomyelitis and spondylodiscitis. The patient recovered completely after a 12-month treatment with combinations of parenteral and oral antibiotics (meropenem, vancomycin, teicoplanin, ciprofloxacin, rifampicin, macrolides etc), including drainage of abscesses and empyema. Eight years after completition of the treatment the patient was without recurrence of R equi infection and lymphoma. Since the eradication od R equi is very difficult, it is very important to make the diagnosis and initiate appropriate antibiotic therapy as soon as possible.

  13. Susceptibility testing of Rhodococcus equi: An interlaboratory test.

    PubMed

    Riesenberg, Anne; Kaspar, Heike; Feßler, Andrea T; Werckenthin, Christiane; Schwarz, Stefan

    2016-10-15

    Due to the importance of antimicrobial susceptibility testing (AST) for veterinary diagnostics, a standardised protocol for AST of Rhodococcus equi by broth microdilution has recently been developed and approved by the Clinical and Laboratory Standards Institute (CLSI). The aim of the present study was to test this protocol in an interlaboratory comparative study for its fitness for use in routine laboratory diagnostics. All of the 18 participating laboratories determined the minimum inhibitory concentrations (MIC) of two R. equi strains against 24 antimicrobial agents. The modal MIC values were determined and the acceptable ranges were set as the modal MIC ±1 dilution step. The R. equi field strain Rh110 showed a slightly better performance than the type strain R. equi ATCC(®) 25729. For the different antimicrobial agents tested, the percentage of MIC values within the acceptable ranges varied from 75.9 to 100% for R. equi ATCC(®) 25729, and from 85.2 to 100% for R. equi Rh110. The most homogeneous MIC results (i.e. modal MIC ±1 dilution step) were obtained for oxacillin and vancomycin, while the most divergent results were seen with cefotaxime and ceftiofur. Using a success rate of at least 80% of the strain-specific MICs being within the acceptable ranges as an arbitrary cut-off, only one of the participating laboratories failed to reach this cut-off value for one of the two R. equi strains. Thus, we consider the new protocol fit for use in routine AST of R. equi. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Saccharification of Cellulose by Recombinant Rhodococcus opacus PD630 Strains

    PubMed Central

    Hetzler, Stephan; Bröker, Daniel

    2013-01-01

    The noncellulolytic actinomycete Rhodococcus opacus strain PD630 is the model oleaginous prokaryote with regard to the accumulation and biosynthesis of lipids, which serve as carbon and energy storage compounds and can account for as much as 87% of the dry mass of the cell in this strain. In order to establish cellulose degradation in R. opacus PD630, we engineered strains that episomally expressed six different cellulase genes from Cellulomonas fimi ATCC 484 (cenABC, cex, cbhA) and Thermobifida fusca DSM43792 (cel6A), thereby enabling R. opacus PD630 to degrade cellulosic substrates to cellobiose. Of all the enzymes tested, five exhibited a cellulase activity toward carboxymethyl cellulose (CMC) and/or microcrystalline cellulose (MCC) as high as 0.313 ± 0.01 U · ml−1, but recombinant strains also hydrolyzed cotton, birch cellulose, copy paper, and wheat straw. Cocultivations of recombinant strains expressing different cellulase genes with MCC as the substrate were carried out to identify an appropriate set of cellulases for efficient hydrolysis of cellulose by R. opacus. Based on these experiments, the multicellulase gene expression plasmid pCellulose was constructed, which enabled R. opacus PD630 to hydrolyze as much as 9.3% ± 0.6% (wt/vol) of the cellulose provided. For the direct production of lipids from birch cellulose, a two-step cocultivation experiment was carried out. In the first step, 20% (wt/vol) of the substrate was hydrolyzed by recombinant strains expressing the whole set of cellulase genes. The second step was performed by a recombinant cellobiose-utilizing strain of R. opacus PD630, which accumulated 15.1% (wt/wt) fatty acids from the cellobiose formed in the first step. PMID:23793636

  15. Metabolism of styrene by Rhodococcus rhodochrous NCIMB 13259.

    PubMed Central

    Warhurst, A M; Clarke, K F; Hill, R A; Holt, R A; Fewson, C A

    1994-01-01

    Rhodococcus rhodochrous NCIMB 13259 grows on styrene, toluene, ethylbenzene, and benzene as sole carbon sources. Simultaneous induction tests with cells grown on styrene or toluene showed high rates of oxygen consumption with toluene cis-glycol and 3-methylcatechol, suggesting the involvement of a cis-glycol pathway. 3-Vinylcatechol accumulated when intact cells were incubated with styrene in the presence of 3-fluorocatechol to inhibit catechol dioxygenase activity. Experiments with 18O2 showed that 3-vinylcatechol was produced following a dioxygenase ring attack. Extracts contained a NAD-dependent cis-glycol dehydrogenase, which converted styrene cis-glycol to 3-vinylcatechol. Both catechol 1,2- and 2,3-dioxygenase activities were present, and these were separated from each other and from the activities of cis-glycol dehydrogenase and 2-hydroxymuconic acid semialdehyde hydrolase by ion-exchange chromatography of extracts. 2-Vinylmuconate accumulated in the growth medium when cells were grown on styrene, apparently as a dead-end product, and extracts contained no detectable muconate cycloisomerase activity. 3-Vinylcatechol was cleaved by catechol 2,3-dioxygenase to give a yellow compound, tentatively identified as 2-hydroxy-6-oxoocta-2,4,7-trienoic acid, and the action of 2-hydroxymuconic acid semialdehyde hydrolase on this produced acrylic acid. A compound with the spectral characteristics of 2-hydroxypenta-2,4-dienoate was produced by the action of 2-hydroxymuconic acid semialdehyde hydrolase on the 2,3-cleavage product of 3-methylcatechol. Extracts were able to transform 2-hydroxypenta-2,4-dienoate and 4-hydroxy-2-oxopentanoate into acetaldehyde and pyruvate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8017910

  16. Mathematic modeling for optimum conditions on aflatoxin B1 degradation by the aerobic bacterium Rhodococcus erythropolis

    USDA-ARS?s Scientific Manuscript database

    Response surface methodology was employed to optimize the degradation conditions of AFB1 by Rhodococcus erythropolis in liquid culture. The most important factors that influence the degradation, as identified by a two-level Plackett-Burman design with 6 variables, were temperature, pH, liquid volume...

  17. Rhodococcus Infection in Solid Organ and Hematopoietic Stem Cell Transplant Recipients1

    PubMed Central

    Ariza-Heredia, Ella J.; Nellore, Anoma; Kotton, Camille N.; Kaul, Daniel R.; Morris, Michele I.; Kelesidis, Theodoros; Shah, Harshal; Park, Seo Young; Nguyen, M. Hong; Razonable, Raymund R.

    2017-01-01

    We conducted a case–control study of 18 US transplant recipients with Rhodococcus infection and 36 matched controls. The predominant types of infection were pneumonia and bacteremia. Diabetes mellitus and recent opportunistic infection were independently associated with disease. Outcomes were generally favorable except for 1 relapse and 1 death. PMID:28221102

  18. Cesium accumulation of Rhodococcus erythropolis CS98 strain immobilized in hydrogel matrices.

    PubMed

    Takei, Takayuki; Yamasaki, Mika; Yoshida, Masahiro

    2014-04-01

    Agarose gels were superior to calcium-alginate gels for immobilizing Rhodococcus erythropolis CS98 strain to remove cesium from water. Suitable incubation time of the immobilized cells in cesium solutions, cell number in the gels and volume ratio of the cesium solution to the gels for efficient cesium removal were identified.

  19. Rhodococcus fascians infection after haematopoietic cell transplantation: not just a plant pathogen?

    PubMed Central

    Austin, Melissa C.; Hallstrand, Teal S.; Hoogestraat, Daniel R.; Balmforth, Gregory; Stephens, Karen; Butler-Wu, Susan

    2016-01-01

    Introduction: Rhodococcus spp. have been implicated in a variety of infections in immunocompromised and immunocompetent hosts. Rhodococcus equi is responsible for the majority of reported cases, but Rhodococcus erythropolis, Rhodococcusgordoniae and Rhodococcusruber infections have been described. There are no prior reports of human infection with Rhodococcus fascians. Case presentation: We describe the unexpected finding of R. fascians in liver lesions incidentally noted at autopsy in an immunosuppressed patient status after bone-marrow transplant for acute lymphoblastic leukaemia who died of unrelated causes (septic shock due to Clostridium difficile colitis). At autopsy, an otherwise unremarkable liver contained several dozen well-demarcated sclerotic-appearing lesions measuring 0.1–0.3 cm in size. The absence of other bacterial or fungal DNA in the setting of histologically visible organisms argues against its presence as a contaminant and raises the consideration that R. fascians represents a human pathogen for the immunocompromised. Conclusion: Whether it represents the sole infectious agent responsible for the miliary lesions or a partially treated co-infection is impossible to determine, but our finding continues to reinforce the importance of molecular techniques in associating organisms with sites of infection and optimizing treatment of infectious diseases. PMID:28348752

  20. Conversion of the Pseudomonas aeruginosa Quinolone Signal and Related Alkylhydroxyquinolines by Rhodococcus sp. Strain BG43

    PubMed Central

    Müller, Christine; Birmes, Franziska S.; Niewerth, Heiko

    2014-01-01

    A bacterial strain, which based on the sequences of its 16S rRNA, gyrB, catA, and qsdA genes, was identified as a Rhodococcus sp. closely related to Rhodococcus erythropolis, was isolated from soil by enrichment on the Pseudomonas quinolone signal [PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone], a quorum sensing signal employed by the opportunistic pathogen Pseudomonas aeruginosa. The isolate, termed Rhodococcus sp. strain BG43, cometabolically degraded PQS and its biosynthetic precursor 2-heptyl-4(1H)-quinolone (HHQ) to anthranilic acid. HHQ degradation was accompanied by transient formation of PQS, and HHQ hydroxylation by cell extracts required NADH, indicating that strain BG43 has a HHQ monooxygenase isofunctional to the biosynthetic enzyme PqsH of P. aeruginosa. The enzymes catalyzing HHQ hydroxylation and PQS degradation were inducible by PQS, suggesting a specific pathway. Remarkably, Rhodococcus sp. BG43 is also capable of transforming 2-heptyl-4-hydroxyquinoline-N-oxide to PQS. It thus converts an antibacterial secondary metabolite of P. aeruginosa to a quorum sensing signal molecule. PMID:25239889

  1. Conversion of the Pseudomonas aeruginosa Quinolone Signal and Related Alkylhydroxyquinolines by Rhodococcus sp. Strain BG43.

    PubMed

    Müller, Christine; Birmes, Franziska S; Niewerth, Heiko; Fetzner, Susanne

    2014-12-01

    A bacterial strain, which based on the sequences of its 16S rRNA, gyrB, catA, and qsdA genes, was identified as a Rhodococcus sp. closely related to Rhodococcus erythropolis, was isolated from soil by enrichment on the Pseudomonas quinolone signal [PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone], a quorum sensing signal employed by the opportunistic pathogen Pseudomonas aeruginosa. The isolate, termed Rhodococcus sp. strain BG43, cometabolically degraded PQS and its biosynthetic precursor 2-heptyl-4(1H)-quinolone (HHQ) to anthranilic acid. HHQ degradation was accompanied by transient formation of PQS, and HHQ hydroxylation by cell extracts required NADH, indicating that strain BG43 has a HHQ monooxygenase isofunctional to the biosynthetic enzyme PqsH of P. aeruginosa. The enzymes catalyzing HHQ hydroxylation and PQS degradation were inducible by PQS, suggesting a specific pathway. Remarkably, Rhodococcus sp. BG43 is also capable of transforming 2-heptyl-4-hydroxyquinoline-N-oxide to PQS. It thus converts an antibacterial secondary metabolite of P. aeruginosa to a quorum sensing signal molecule.

  2. Comparative and Functional Genomics of Rhodococcus opacus PD630 for Biofuels Development

    PubMed Central

    Holder, Jason W.; Ulrich, Jil C.; DeBono, Anthony C.; Godfrey, Paul A.; Desjardins, Christopher A.; Zucker, Jeremy; Zeng, Qiandong; Leach, Alex L. B.; Ghiviriga, Ion; Dancel, Christine; Abeel, Thomas; Gevers, Dirk; Kodira, Chinnappa D.; Desany, Brian; Affourtit, Jason P.; Birren, Bruce W.; Sinskey, Anthony J.

    2011-01-01

    The Actinomycetales bacteria Rhodococcus opacus PD630 and Rhodococcus jostii RHA1 bioconvert a diverse range of organic substrates through lipid biosynthesis into large quantities of energy-rich triacylglycerols (TAGs). To describe the genetic basis of the Rhodococcus oleaginous metabolism, we sequenced and performed comparative analysis of the 9.27 Mb R. opacus PD630 genome. Metabolic-reconstruction assigned 2017 enzymatic reactions to the 8632 R. opacus PD630 genes we identified. Of these, 261 genes were implicated in the R. opacus PD630 TAGs cycle by metabolic reconstruction and gene family analysis. Rhodococcus synthesizes uncommon straight-chain odd-carbon fatty acids in high abundance and stores them as TAGs. We have identified these to be pentadecanoic, heptadecanoic, and cis-heptadecenoic acids. To identify bioconversion pathways, we screened R. opacus PD630, R. jostii RHA1, Ralstonia eutropha H16, and C. glutamicum 13032 for growth on 190 compounds. The results of the catabolic screen, phylogenetic analysis of the TAGs cycle enzymes, and metabolic product characterizations were integrated into a working model of prokaryotic oleaginy. PMID:21931557

  3. Analysis of genome sequences from plant pathogenic Rhodococcus reveals genetic novelties in virulence loci

    USDA-ARS?s Scientific Manuscript database

    Members of Gram-positive Actinobacteria cause economically important diseases to plants. Within the Rhodococcus genus, some members can cause growth deformities and persist as pathogens on a wide range of host plants. The current model predicts that phytopathogenic isolates require a cluster of thre...

  4. Whole-Genome Shotgun Sequence of Rhodococcus Species Strain JVH1

    PubMed Central

    Brooks, Shannon L.

    2012-01-01

    Here we present a whole-genome shotgun sequence of Rhodococcus species strain JVH1, an organism capable of degrading a variety of organosulfur compounds. In particular, JVH1 is able to selectively cleave carbon-sulfur bonds within alkyl chains. A large number of oxygenases were identified, consistent with other members of the genus. PMID:22965106

  5. Complete Genome Sequence of Rhodococcus sp. Strain IcdP1 Shows Diverse Catabolic Potential

    PubMed Central

    Qu, Jie; Miao, Li-Li; Liu, Ying

    2015-01-01

    The complete genome sequence of Rhodococcus sp. strain IcdP1 is presented here. This organism was shown to degrade a broad range of high-molecular-weight polycyclic aromatic hydrocarbons and organochlorine pesticides. The sequence data can be used to predict genes for xenobiotic biodegradation and metabolism. PMID:26139718

  6. First report of sepsis caused by Rhodococcus corynebacterioides in a patient with myelodysplastic syndrome.

    PubMed

    Kitamura, Yuka; Sawabe, Etsuko; Ohkusu, Kiyofumi; Tojo, Naoko; Tohda, Shuji

    2012-03-01

    We report a case of sepsis caused by Rhodococcus corynebacterioides, identified using 16S rRNA gene sequencing, in a myelodysplastic syndrome patient who had undergone hematopoietic stem cell transplantation. This is the first report of R. corynebacterioides infection in a human.

  7. First Report of Sepsis Caused by Rhodococcus corynebacterioides in a Patient with Myelodysplastic Syndrome

    PubMed Central

    Kitamura, Yuka; Sawabe, Etsuko; Ohkusu, Kiyofumi; Tojo, Naoko

    2012-01-01

    We report a case of sepsis caused by Rhodococcus corynebacterioides, identified using 16S rRNA gene sequencing, in a myelodysplastic syndrome patient who had undergone hematopoietic stem cell transplantation. This is the first report of R. corynebacterioides infection in a human. PMID:22205796

  8. Complete Genome Sequence of Rhodococcus sp. Strain WMMA185, a Marine Sponge-Associated Bacterium

    PubMed Central

    Adnani, Navid; Braun, Doug R.; McDonald, Bradon R.; Chevrette, Marc G.; Currie, Cameron R.

    2016-01-01

    The Rhodococcus strain WMMA185 was isolated from the marine sponge Chondrilla nucula as part of ongoing drug discovery efforts. Analysis of the 4.44-Mb genome provides information regarding interspecies interactions as pertains to regulation of secondary metabolism and natural product biosynthetic potentials. PMID:27979952

  9. Complete Genome Sequence of the Polychlorinated Biphenyl Degrader Rhodococcus sp. WB1

    PubMed Central

    Yu, Man; Shen, Alin

    2016-01-01

    Rhodococcus sp. WB1 is a polychlorinated biphenyl degrader which was isolated from contaminated soil in Zhejiang, China. Here, we present the complete genome sequence. The analysis of this genome indicated that a biphenyl-degrading gene cluster and several xenobiotic metabolism pathways are harbored. PMID:27738025

  10. Rhodococcus equi hyperimmune plasma decreases pneumonia severity after a randomised experimental challenge of neonatal foals.

    PubMed

    Sanz, M G; Loynachan, A; Horohov, D W

    2016-03-12

    Since a vaccine is not available against Rhodococcus equi, R equi-specific hyperimmune plasma (HIP) is commonly used, although its efficacy remains controversial. The objective of this study was to evaluate the ability of a commercially available HIP to prevent clinical rhodococcal pneumonia in neonatal foals after experimental challenge. British Veterinary Association.

  11. Analysis of Genome Sequences from Plant Pathogenic Rhodococcus Reveals Genetic Novelties in Virulence Loci

    PubMed Central

    Davis, Edward W.; Putnam, Melodie L.; Hu, Erdong; Swader-Hines, David; Mol, Adeline; Baucher, Marie; Prinsen, Els; Zdanowska, Magdalena; Givan, Scott A.; Jaziri, Mondher El; Loper, Joyce E.; Mahmud, Taifo; Chang, Jeff H.

    2014-01-01

    Members of Gram-positive Actinobacteria cause economically important diseases to plants. Within the Rhodococcus genus, some members can cause growth deformities and persist as pathogens on a wide range of host plants. The current model predicts that phytopathogenic isolates require a cluster of three loci present on a linear plasmid, with the fas operon central to virulence. The Fas proteins synthesize, modify, and activate a mixture of growth regulating cytokinins, which cause a hormonal imbalance in plants, resulting in abnormal growth. We sequenced and compared the genomes of 20 isolates of Rhodococcus to gain insights into the mechanisms and evolution of virulence in these bacteria. Horizontal gene transfer was identified as critical but limited in the scale of virulence evolution, as few loci are conserved and exclusive to phytopathogenic isolates. Although the fas operon is present in most phytopathogenic isolates, it is absent from phytopathogenic isolate A21d2. Instead, this isolate has a horizontally acquired gene chimera that encodes a novel fusion protein with isopentyltransferase and phosphoribohydrolase domains, predicted to be capable of catalyzing and activating cytokinins, respectively. Cytokinin profiling of the archetypal D188 isolate revealed only one activate cytokinin type that was specifically synthesized in a fas-dependent manner. These results suggest that only the isopentenyladenine cytokinin type is synthesized and necessary for Rhodococcus phytopathogenicity, which is not consistent with the extant model stating that a mixture of cytokinins is necessary for Rhodococcus to cause leafy gall symptoms. In all, data indicate that only four horizontally acquired functions are sufficient to confer the trait of phytopathogenicity to members of the genetically diverse clade of Rhodococcus. PMID:25010934

  12. Isolation and characterization of RDX-degrading Rhodococcus species from a contaminated aquifer.

    PubMed

    Bernstein, Anat; Adar, Eilon; Nejidat, Ali; Ronen, Zeev

    2011-09-01

    Groundwater contamination by the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a global problem. Israel's coastal aquifer was contaminated with RDX. This aquifer is mostly aerobic and we therefore sought aerobic bacteria that might be involved in natural attenuation of the compound in the aquifer. RDX-degrading bacteria were captured by passively sampling the indigenous bacteria onto sterile sediments placed within sampling boreholes. Aerobic RDX biodegradation potential was detected in the sediments sampled from different locations along the plume. RDX degradation with the native sampled consortium was accompanied by 4-nitro-2,4-diazabutanal formation. Two bacterial strains of the genus Rhodococcus were isolated from the sediments and identified as aerobic RDX degraders. The xplA gene encoding the cytochrome P450 enzyme was partially (~500 bp) sequenced from both isolates. The obtained DNA sequences had 99% identity with corresponding gene fragments of previously isolated RDX-degrading Rhodococcus strains. RDX degradation by both strains was prevented by 200 μM of the cytochrome P450 inhibitor metyrapone, suggesting that cytochrome P450 indeed mediates the initial step in RDX degradation. RDX biodegradation activity by the T7 isolate was inhibited in the presence of nitrate or ammonium concentrations above 1.6 and 5.5 mM, respectively (100 mg l(-1)) while the T9N isolate's activity was retarded only by ammonium concentrations above 5.5 mM. This study shows that bacteria from the genus Rhodococcus, potentially degrade RDX in the saturated zone as well, following the same aerobic degradation pathway defined for other Rhodococcus species. RDX-degrading activity by the Rhodococcus species isolate T9N may have important implications for the bioremediation of nitrate-rich RDX-contaminated aquifers.

  13. Rhodococcus canchipurensis sp. nov., an actinomycete isolated from a limestone deposit site.

    PubMed

    Nimaichand, Salam; Sanasam, Suchitra; Zheng, Liu-Qiang; Zhu, Wen-Yong; Yang, Ling-Ling; Tang, Shu-Kun; Ningthoujam, Debananda S; Li, Wen-Jun

    2013-01-01

    A novel actinobacterial strain, MBRL 353(T), was isolated from a sample collected from a limestone quarry at Hundung, Manipur, India. Comparison of 16S rRNA gene sequences of strain MBRL 353(T) and other members of the genus Rhodococcus showed sequence similarities ranging from 95.5 to 98.2 %, with strain MBRL 353(T) showing closest sequence similarity to Rhodococcus triatomae IMMIB RIV-085(T) (98.2 %) and Rhodococcus equi DSM 20307(T) (97.2 %). DNA-DNA hybridization results, however, revealed that DNA-DNA relatedness values between strain MBRL 353(T) and R. triatomae DSM 44892(T) (43.4 %) and R. equi DSM 20307(T) (33.4 %) were well below the 70 % limit for species identification. Strain MBRL 353(T) contained meso-diaminopimelic acid as the diagnostic diamino acid and galactose and arabinose in the cell wall. Mycolic acids were present. The major fatty acids were C(16 : 0) (45.7 %), C(18 : 1)ω9c (18.2 %) and 10-methyl C(18 : 0) (11.3 %). The only menaquinone detected was MK-8(H(2)), while the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and one unknown phospholipid. The G+C content of the genomic DNA was 69.2 mol%. The phenotypic and genotypic data showed that strain MBRL 353(T) merits recognition as a representative of a novel species of the genus Rhodococcus for which the name Rhodococcus canchipurensis sp. nov. is proposed; the type strain is MBRL 353(T) (= KCTC 19851(T) = JCM 17578(T)).

  14. Draft Genome Sequence of Rhodococcus erythropolis NSX2, an Actinobacterium Isolated from a Cadmium-Contaminated Environment

    PubMed Central

    Egidi, Eleonora; Wood, Jennifer L.; Fox, Edward M.; Liu, Wuxing

    2016-01-01

    Rhodococcus erythropolis NSX2 is a rhizobacterium isolated from a heavy metal–contaminated environment. The 6.2-Mb annotated genome sequence shows that this strain harbors genes associated with heavy-metal resistance and xenobiotics degradation. PMID:27795276

  15. [Cloning of new acylamidase gene from Rhodococcus erythropolis and its expression in Escherichia coli].

    PubMed

    2013-10-01

    The gene for new Rhodococcus erythropolis TA37 acylamidase, which possesses unique substrate specificity, has been cloned and expressed in E. coli. Substrates for this enzyme are not only simple amides, such as acetamide and propionamide, but also N-substituted amides, such as 4'-nitroacetanilide. The 1431-bp gene was expressed in E. coli BL21 (DE3) cells on pET16b plasmid under the control of a promoter of the φ 10 gene from the T7 phage. The molecular mass of recombinant acylamidase in E. coli was 55 kDa, which corresponded to that of native acylamidase from Rhodococcus erythropolis TA37. Recombinant acylamidase was able to hydrolize N-substituted amides. A search of a nucleotide database and multiple alignment revealed that acylamidase belonged to the Amidase protein family PF01425, but its nucleotide and amino acid sequences differed significantly from those of the described amidases.

  16. Rhodococcus equi pericarditis in a patient living with HIV/AIDS.

    PubMed

    Gundelly, Praveen; Thornton, Alice; Greenberg, Richard N; McCormick, Malkanthie; Myint, Thein

    2014-01-01

    Rhodococcus equi, previously called Corynebacterium equi, is known to cause pneumonia in foals and swine. Although it was known to cause infection rarely in humans, R equi infection in humans has increased with the advent of HIV and increased use of immunosuppressants. We report a case of a 48-year-old male patient with newly diagnosed HIV/AIDS presenting with recurrent R equi bacteremia, pericardial effusion, and pericardial cyst. The infection was treated with drainage of the pericardial effusion and cyst and 2 weeks of intravenous vancomycin and 6 months of oral azithromycin and levofloxacin. Rhodococcus equi causes pericarditis and pericardial effusion. It can be effectively treated with debridement, drainage, and a prolonged course of antibiotics. In vitro antibiotic susceptibility should be checked as resistance to antibiotics can develop, especially if drainage is inadequate.

  17. A novel lipopeptide produced by a Pacific Ocean deep-sea bacterium, Rhodococcus sp. TW53.

    PubMed

    Peng, F; Wang, Y; Sun, F; Liu, Z; Lai, Q; Shao, Z

    2008-09-01

    Our goal was to find a novel, biosurfactant-producing bacterium from Pacific Ocean deep-sea sediments. An oil-degrading biosurfactant-producing bacterium TW53 was obtained from deep-sea sediment, and was identified through 16S rDNA analysis as belonging to the genus Rhodococcus. It lowered the surface tension of its culture to 34.4 mN m(-1). Thin layer chromatography (TLC) showed that the crude biosurfactants of TW53 were composed of lipopeptides and free fatty acids (FA). The lipopeptides were purified with column chromatography and then hydrolysed with 6 mol l(-1) HCl. Gas chromatography-mass spectrometry analysis showed that the hydrolyte in the hydrophobic fraction contained five kinds of FA with chain lengths of C(14)-C(19), and C(16)H(32)O(2) was a major component making up 59.18% of the total. However, 3-hydroxyl FA was not found, although it is usually found in lipopeptides. Silica gel TLC revealed that the hydrolyte in the hydrophilic fraction was composed of five kinds of amino acids; consistently, ESI-Q-TOF-MS analysis confirmed the composition results and provided their sequence tentatively as Ala-Ile-Asp-Met-Pro. Furthermore, the yield and CMC (critical micelle concentrations) of purified lipopeptides were examined. The purified product reduced the surface tension of water to 30.7 mN m(-1) with a CMC value of 23.7 mg l(-1). These results suggest that Rhodococcus sp. TW53 produces a novel lipopeptide that we have named rhodofactin. The deep-sea isolate Rhodococcus sp. TW53 was the first reported lipopeptide-producing bacterium of this genus. The lipopeptides had novel chemical compositions. Rhodococcus sp. TW53 has potential in the exploration of new biosurfactants and could be used in bioremediation of marine oil pollution.

  18. Minimum inhibitory concentrations of erythromycin and rifampin for Rhodococcus equi during the years 2007-2014.

    PubMed

    Fenton, Caitriona S; Buckley, Thomas C

    2015-01-01

    Rhodococcus equi is a gram positive, intracellular pathogen of foals worldwide. The aim of this study was to determine whether there was an increasing resistance occurring in Rhodococcus equi towards the antibiotics rifampin and erythromycin over a seven year period. The investigation was carried out with the use of E test strips (epsilometers) for rifampin and erythromycin in order to determine the Minimum Inhibitory Concentrations (MIC) values of Rhodococcus equi to these antibiotics. The main results of this study found that the mean MICs were higher for erythromycin than for rifampin for every year analysed apart from 2008. The results highlight that 75 % (6/8) of the mean MICs for erythromycin were above the threshold of susceptibility of 0.5 μg/ml and one of the yearly mean MICs for rifampin (2008) was above the level of ≤ 1 μg/ml. Two soil samples analysed had high MIC values of 2 μg/ml and 3 μg/ml for rifampin and erythromycin respectively. These samples can be said to have acquired resistance as they are above 1 μg/ml. The significance of these findings is that R. equi is already a problematic pathogen to treat and if the bacteria keeps gaining resistance to these antibiotics at rate that has been shown over the last decade, then a new form of treatment will have to be introduced. Further research into the genomics of Rhodococcus equi will, in time, shed more light on possible alternatives such as vaccines or new, more effective antimicrobials.

  19. Activity of Clarithromycin or Rifampin Alone or in Combination against Experimental Rhodococcus equi Infection in Mice

    PubMed Central

    Burton, Alexandra J.; Berghaus, Londa J.; Hondalus, Mary K.

    2015-01-01

    Treatment of mice with the combination of clarithromycin with rifampin resulted in a significantly lower number of Rhodococcus equi CFU in the organs of mice than treatment with either drug alone or placebo. There was no significant difference in the number of R. equi CFU between mice treated with clarithromycin monotherapy, rifampin monotherapy, or placebo. The combination of clarithromycin with rifampin conferred a clear advantage over either drug as monotherapy in this model of chronic R. equi infection. PMID:25824218

  20. Biodegradation and metabolic pathway of nicotine in Rhodococcus sp. Y22.

    PubMed

    Gong, Xiaowei; Ma, Guanghui; Duan, Yanqing; Zhu, Donglai; Chen, Yongkuan; Zhang, Ke-Qin; Yang, Jinkui

    2016-11-01

    Nicotine in tobacco is harmful to health and the environment, so there is an environmental requirement to remove nicotine from tobacco and tobacco wastes. In this study, the biotransformation of nicotine by Rhodococcus sp. Y22 was investigated, and three metabolites (NIC1, NIC4 and NIC5) were isolated by column separation, preparative TLC and solid plate's method, respectively. NIC1 was identified as 6-hydoxynicotine based on the results of NMR, MS, HPLC-UV and HRESIMS analysis; NIC4 was a novel compound and identified as 5-(3-methyl-[1,3]oxazinan-2-ylidene)-5H-pyridin-2-one based on the results of NMR, MS and UV analysis; NIC5 was identified as nicotine blue based on the results of NMR and MS analysis. Meanwhile, two metabolites NIC2 and NIC3 were identified as 6-hydroxy-N-methylmyosmine and 6-hydroxypseudooxynicotine by HRESIMS analysis, respectively. According to these metabolites, the possible pathway of nicotine degradation by Rhodococcus sp. Y22 was proposed. The nicotine can be transformed to nicotine blue through two pathways (A and B), and 6-hydroxy-N-methylmyosmine is the key compound, which can be converted to 6-hydroxypseudooxynicotine (pathway A) and 5-(3-methyl-[1,3]oxazinan-2-ylidene)-5H-pyridin-2-one (pathway B), respectively. Moreover, the encoding gene of nicotine dehydrogenase, ndh, was amplified from Rhodococcus sp. Y22, and its transcriptional level could be up-regulated obviously under nicotine induction. Our studies reported the key metabolites and possible biotransformation pathway of nicotine in Rhodococcus sp. Y22, and provided new insights into the microbial metabolism of nicotine.

  1. Detection, isolation and partial characterization of an immunostimulating glycoprotein from Rhodococcus fascians.

    PubMed

    Butschak, Günter; Karsten, Uwe; Schelhaas, Ute; Ott, Holger; Emmendörffer, Andreas; Niemeyer, Bernd; Helmholz, Heike; Goletz, Steffen

    2006-09-01

    In a search for novel immunostimulating substances we detected that culture supernatants of the gram-positive phytopathogenic bacterium, Rhodococcus fascians, were able to induce cytokine release (TNF(alpha)) from mouse peritoneal macrophages. Monoclonal antibodies were generated against the active principle, and were employed for its isolation and partial characterization as a high molecular (MW>100 kDa) glycoprotein. In addition, methods practicable for its biotechnological preparation and several ELISA variants for its determination were developed.

  2. Metabolism of naphthalene, 1-naphthol, indene, and indole by Rhodococcus sp. strain NCIMB 12038

    SciTech Connect

    Allen, C.C.R.; Boyd, D.R.; Larkin, M.J.

    1997-01-01

    Naphthalene and its 1-hydroxy derivative, 1-naphthol, are examples of common bicyclic aromatics often released into the environment. Bacteria which degrade these compounds under aerobic conditions are widely distributed; however, the metabolism of naphthalene in gram-positive nocardioform bacteria has not been well investigated. This study described the independent regulation of 1-naphthol and naphthalene metabolism in Rhodococcus strain 12038. 45 refs., 2 figs., 1 tab.

  3. Isolation and Characterization of o-Xylene Oxygenase Genes from Rhodococcus opacus TKN14

    PubMed Central

    Maruyama, Takahiro; Ishikura, Masaharu; Taki, Hironori; Shindo, Kazutoshi; Kasai, Hiroaki; Haga, Miyuki; Inomata, Yukie; Misawa, Norihiko

    2005-01-01

    o-Xylene is one of the most difficult-to-degrade environmental pollutants. We report here Rhodococcus genes mediating oxygenation in the first step of o-xylene degradation. Rhodococcus opacus TKN14, isolated from soil contaminated with o-xylene, was able to utilize o-xylene as the sole carbon source and to metabolize it to o-methylbenzoic acid. A cosmid library from the genome of this strain was constructed in Escherichia coli. A bioconversion analysis revealed that a cosmid clone incorporating a 15-kb NotI fragment had the ability to convert o-xylene into o-methylbenzyl alcohol. The sequence analysis of this 15-kb region indicated the presence of a gene cluster significantly homologous to the naphthalene-inducible dioxygenase gene clusters (nidABCD) that had been isolated from Rhodococcus sp. strain I24. Complementation studies, using E. coli expressing various combinations of individual open reading frames, revealed that a gene (named nidE) for rubredoxin (Rd) and a novel gene (named nidF) encoding an auxiliary protein, which had no overall homology with any other proteins, were indispensable for the methyl oxidation reaction of o-xylene, in addition to the dioxygenase iron-sulfur protein genes (nidAB). Regardless of the presence of NidF, the enzyme composed of NidABE was found to function as a typical naphthalene dioxygenase for converting naphthalene and various (di)methylnaphthalenes into their corresponding cis-dihydrodiols. All the nidABEF genes were transcriptionally induced in R. opacus TKN14 by the addition of o-xylene to a mineral salt medium. It is very likely that these genes are involved in the degradation pathways of a wide range of aromatic hydrocarbons by Rhodococcus species as the first key enzyme. PMID:16332743

  4. Isolation and characterization of o-xylene oxygenase genes from Rhodococcus opacus TKN14.

    PubMed

    Maruyama, Takahiro; Ishikura, Masaharu; Taki, Hironori; Shindo, Kazutoshi; Kasai, Hiroaki; Haga, Miyuki; Inomata, Yukie; Misawa, Norihiko

    2005-12-01

    o-Xylene is one of the most difficult-to-degrade environmental pollutants. We report here Rhodococcus genes mediating oxygenation in the first step of o-xylene degradation. Rhodococcus opacus TKN14, isolated from soil contaminated with o-xylene, was able to utilize o-xylene as the sole carbon source and to metabolize it to o-methylbenzoic acid. A cosmid library from the genome of this strain was constructed in Escherichia coli. A bioconversion analysis revealed that a cosmid clone incorporating a 15-kb NotI fragment had the ability to convert o-xylene into o-methylbenzyl alcohol. The sequence analysis of this 15-kb region indicated the presence of a gene cluster significantly homologous to the naphthalene-inducible dioxygenase gene clusters (nidABCD) that had been isolated from Rhodococcus sp. strain I24. Complementation studies, using E. coli expressing various combinations of individual open reading frames, revealed that a gene (named nidE) for rubredoxin (Rd) and a novel gene (named nidF) encoding an auxiliary protein, which had no overall homology with any other proteins, were indispensable for the methyl oxidation reaction of o-xylene, in addition to the dioxygenase iron-sulfur protein genes (nidAB). Regardless of the presence of NidF, the enzyme composed of NidABE was found to function as a typical naphthalene dioxygenase for converting naphthalene and various (di)methylnaphthalenes into their corresponding cis-dihydrodiols. All the nidABEF genes were transcriptionally induced in R. opacus TKN14 by the addition of o-xylene to a mineral salt medium. It is very likely that these genes are involved in the degradation pathways of a wide range of aromatic hydrocarbons by Rhodococcus species as the first key enzyme.

  5. The detection and phylogenetic analysis of the alkane 1-monooxygenase gene of members of the genus Rhodococcus.

    PubMed

    Táncsics, András; Benedek, Tibor; Szoboszlay, Sándor; Veres, Péter G; Farkas, Milán; Máthé, István; Márialigeti, Károly; Kukolya, József; Lányi, Szabolcs; Kriszt, Balázs

    2015-02-01

    Naturally occurring and anthropogenic petroleum hydrocarbons are potential carbon sources for many bacteria. The AlkB-related alkane hydroxylases, which are integral membrane non-heme iron enzymes, play a key role in the microbial degradation of many of these hydrocarbons. Several members of the genus Rhodococcus are well-known alkane degraders and are known to harbor multiple alkB genes encoding for different alkane 1-monooxygenases. In the present study, 48 Rhodococcus strains, representing 35 species of the genus, were investigated to find out whether there was a dominant type of alkB gene widespread among species of the genus that could be used as a phylogenetic marker. Phylogenetic analysis of rhodococcal alkB gene sequences indicated that a certain type of alkB gene was present in almost every member of the genus Rhodococcus. These alkB genes were common in a unique nucleotide sequence stretch absent from other types of rhodococcal alkB genes that encoded a conserved amino acid motif: WLG(I/V/L)D(G/D)GL. The sequence identity of the targeted alkB gene in Rhodococcus ranged from 78.5 to 99.2% and showed higher nucleotide sequence variation at the inter-species level compared to the 16S rRNA gene (93.9-99.8%). The results indicated that the alkB gene type investigated might be applicable for: (i) differentiating closely related Rhodococcus species, (ii) properly assigning environmental isolates to existing Rhodococcus species, and finally (iii) assessing whether a new Rhodococcus isolate represents a novel species of the genus. Copyright © 2014 Elsevier GmbH. All rights reserved.

  6. Seroprevalence of Rhodococcus equi in horses in Israel.

    PubMed

    Tirosh-Levy, Sharon; Gürbilek, Sevil E; Tel, Osman Y; Keskin, Oktay; Steinman, Amir

    2017-06-26

    Rhodococcus equi is a common cause of pneumonia in foals and has extensive clinical, economic and possibly zoonotic consequences. This bacterium survives well in the environment and may be considered as normal flora of adult horses. Certain strains of this bacterium are extremely virulent in foals, and early identification and intervention is crucial for prognosis. Rhodococcus equi is endemic in many parts of the world and occasionally isolated in Israel. This study was designed to evaluate R. equi seroprevalence in adult horses in Israel to indirectly indicate the potential level of exposure of susceptible foals. Sera were collected from 144 horses during spring 2011 and from 293 horses during fall 2014, and the presence of antibodies against virulent R. equi was detected by enzyme-linked immunosorbent assay. Equine seroprevalence of R. equi was found to be 7.6% in 2011 and 5.1% in 2014. Only one farm had seropositive horses in 2011, whereas several farms had seropositive horses in 2014. No significant risk factors for seropositivity were found. Rhodococcus equi appears to be endemic in Israel. This is the first survey of R. equi in Israel that provides information on the epidemiology of this important bacterium.

  7. Microbial biodiesel production from oil palm biomass hydrolysate using marine Rhodococcus sp. YHY01.

    PubMed

    Bhatia, Shashi Kant; Kim, Junyoung; Song, Hun-Seok; Kim, Hyun Joong; Jeon, Jong-Min; Sathiyanarayanan, Ganesan; Yoon, Jeong-Jun; Park, Kyungmoon; Kim, Yun-Gon; Yang, Yung-Hun

    2017-02-20

    The effect of various biomass derived inhibitors (i.e. furfural, hydroxymethylfurfural (HMF), vanillin, 4-hydroxy benzaldehyde (4-HB) and acetate) was investigated for fatty acid accumulation in Rhodococcus sp. YHY 01. Rhodococcus sp. YHY01 was able to utilize acetate, vanillin, and 4-HB for biomass production and fatty acid accumulation. The IC50 value for furfural (3.1mM), HMF (3.2mM), vanillin (2.0mM), 4-HB (2.7mM) and acetate (3.7mM) was calculated. HMF and vanillin affect fatty acid composition and increase saturated fatty acid content. Rhodococcus sp. YHY 01 cultured with empty fruit bunch hydrolysate (EFBH) as the main carbon source resulted in enhanced biomass (20%) and fatty acid productivity (37%), in compression to glucose as a carbon source. Overall, this study showed the beneficial effects of inhibitory molecules on growth and fatty acid production, and support the idea of biomass hydrolysate utilization for biodiesel production by avoiding complex efforts to remove inhibitory compounds.

  8. Biofouling inhibition in MBR by Rhodococcus sp. BH4 isolated from real MBR plant.

    PubMed

    Oh, Hyun-Suk; Kim, Sang-Ryoung; Cheong, Won-Suk; Lee, Chung-Hak; Lee, Jung-Kee

    2013-12-01

    It has been reported that an indigenous quorum quenching bacterium, Rhodococcus sp. BH4, which was isolated from a real plant of membrane bioreactor (MBR) has promising potential to control biofouling in MBR. However, little is known about quorum quenching mechanisms by the strain BH4. In this study, various characteristics of strain BH4 were investigated to elucidate its behavior in more detail in the mixed liquor of MBR. The N-acyl homoserine lactone hydrolase (AHL-lactonase) gene of strain BH4 showed a high degree of identity to qsdA in Rhodococcus erythropolis W2. The LC-ESI-MS analysis of the degradation product by strain BH4 confirmed that it inactivated AHL activity by hydrolyzing the lactone bond of AHL. It degraded a wide range of N-acyl homoserine lactones (AHLs), but there was a large difference in the degradation rate of each AHL compared to other reported AHL-lactonase-producing strains belonging to Rhodococcus genus. Its quorum quenching activity was confirmed not only in the Luria-Bertani medium, but also in the synthetic wastewater. Furthermore, the amount of strain BH4 encapsulated in the vessel as well as the material of the vessel substantially affected the quorum quenching activity of strain BH4, which provides useful information, particularly for the biofouling control in a real MBR plant from an engineering point of view.

  9. Membrane transport systems and the biodegradation potential and pathogenicity of genus Rhodococcus

    PubMed Central

    de Carvalho, Carla C. C. R.; Costa, Sofia S.; Fernandes, Pedro; Couto, Isabel; Viveiros, Miguel

    2014-01-01

    The Rhodococcus genus contains species with remarkable ability to tolerate toxic compounds and to degrade a myriad of substrates. These substrates have to cross a distinctive cell envelope dominated by mycolic acids anchored in a scaffold of arabinogalactan covalently attached to the cell wall peptidoglycan, and a cellular membrane with phospholipids, whose composition in fatty acids can be rapidly altered in response to environmental conditions. The hydrophobic nature of the cell envelope facilitates the entrance of hydrophobic molecules but some substrates require active transport systems. Additionally, toxic compounds may also be extruded by energy spending efflux systems. In this review, physiological evidences of the use of transport systems by Rhodococcus strains and genomic studies that corroborate their existence are presented and discussed. The recently released complete genomes of several Rhodococcus strains will be the basis for an in silico correlation analysis between the efflux pumps present in the genome and their role on active transport of substrates. These transport systems will be placed on an integrative perspective of the impact of this important genus on biotechnology and health, ranging from bioremediation to antibiotic and biocide resistance. PMID:24772091

  10. Cometabolic Degradation of Trichloroethene by Rhodococcus sp. Strain L4 Immobilized on Plant Materials Rich in Essential Oils▿ †

    PubMed Central

    Suttinun, Oramas; Müller, Rudolf; Luepromchai, Ekawan

    2010-01-01

    The cometabolic degradation of trichloroethene (TCE) by Rhodococcus sp. L4 was limited by the loss of enzyme activity during TCE transformation. This problem was overcome by repeated addition of inducing substrates, such as cumene, limonene, or cumin aldehyde, to the cells. Alternatively, Rhodococcus sp. L4 was immobilized on plant materials which contain those inducers in their essential oils. Cumin seeds were the most suitable immobilizing material, and the immobilized cells tolerated up to 68 μM TCE and degraded TCE continuously. The activity of immobilized cells, which had been inactivated partially during TCE degradation, could be reactivated by incubation in mineral salts medium without TCE. These findings demonstrate that immobilization of Rhodococcus sp. L4 on plant materials rich in essential oils is a promising method for efficient cometabolic degradation of TCE. PMID:20472723

  11. Structural analysis of the 6 kb cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 and construction of Escherichia coli-Rhodococcus shuttle vectors.

    PubMed

    De Mot, R; Nagy, I; De Schrijver, A; Pattanapipitpaisal, P; Schoofs, G; Vanderleyden, J

    1997-10-01

    The complete nucleotide sequence of the 5936 bp cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 was determined. Based on the characteristics of its putative replication genes, repA and repB, pFAJ2600 was assigned to the family of pAL5000-related small replicons identified in Mycobacterium (pAL5000), Corynebacterium (pXZ10142), Brevibacterium (pRBL1), Bifidobacterium (pMB1) and Neisseria (pJD1). The replication systems of these plasmids show striking similarities to the ones used by the ColE2 family of plasmids from Enterobacteria with respect to both trans-acting factors and ori sequences. Two possible plasmid stabilization systems are encoded on pFAJ2600: a site-specific recombinase (PmrA) related to the Escherichia coli Xer proteins for plasmid multimer resolution and an ATPase (ParA) related to the A-type of proteins in sop/par partitioning systems. The proposed replication termination region of pFAJ2600 has features in common with the Ter loci of Bacillus subtilis. Chimeras composed of a pUC18-Cmr derivative inserted in the parA-repA intergenic region of vector pFAJ2600 produced vectors that could be shuttled between Escherichia coli and several Rhodococcus species (R. erythropolis, R. fascians, R. rhodochrous, R. ruber). The pFAJ2600-based shuttle vector pFAJ2574 was stably maintained in R. erythropolis and R. fascians growing under non-selective conditions.

  12. Rhodococcus opacus B4: a promising bacterium for production of biofuels and biobased chemicals.

    PubMed

    Castro, Ana Rita; Rocha, Isabel; Alves, Maria Madalena; Pereira, Maria Alcina

    2016-12-01

    Bacterial lipids have relevant applications in the production of renewable fuels and biobased oleochemicals. The genus Rhodococcus is one of the most relevant lipid producers due to its capability to accumulate those compounds, mainly triacylglycerols (TAG), when cultivated on different defined substrates, namely sugars, organic acids and hydrocarbons but also on complex carbon sources present in industrial wastes. In this work, the production of storage lipids by Rhodococcus opacus B4 using glucose, acetate and hexadecane is reported for the first time and its productivity compared with Rhodococcus opacus PD630, the best TAG producer bacterium reported. Both strains accumulated mainly TAG from all carbon sources, being influenced by the carbon source itself and by the duration of the accumulation period. R. opacus B4 produced 0.09 and 0.14 g L(-1) at 24 and 72 h, with hexadecane as carbon source, which was 2 and 3.3 fold higher than the volumetric production obtained by R. opacus PD630. Both strains presented similar fatty acids (FA) profiles in intact cells while in TAG produced fraction, R. opacus B4 revealed a higher variability in fatty acid composition than R. opacus PD630, when both strains were cultivated on hexadecane. The obtained results open new perspectives for the use of R. opacus B4 to produce TAG, in particular using oily (alkane-contaminated) waste and wastewater as cheap raw-materials. Combining TAG production with hydrocarbons degradation is a promising strategy to achieve environmental remediation while producing added value compounds.

  13. Biodegradation of cyanide by acetonitrile-induced cells of Rhodococcus sp. UKMP-5M.

    PubMed

    Nallapan Maniyam, Maegala; Sjahrir, Fridelina; Ibrahim, Abdul Latif; Cass, Anthony E G

    2013-01-01

    A Rhodococcus sp. UKMP-5M isolate was shown to detoxify cyanide successfully, suggesting the presence of an intrinsic property in the bacterium which required no prior cyanide exposure for induction of this property. However, in order to promote growth, Rhodococcus sp. UKMP-5M was fully acclimatized to cyanide after 7 successive subcultures in 0.1 mM KCN for 30 days. To further shorten the lag phase and simultaneously increase the tolerance towards higher cyanide concentrations, the bacterium was induced with various nitrile compounds sharing a similar degradatory pathway to cyanide. Acetonitrile emerged as the most favored inducer and the induced cells were able to degrade 0.1 mM KCN almost completely within 18 h. With the addition of subsequent aliquots of 0.1 mM KCN a shorter period for complete removal of cyanide was required, which proved to be advantageous economically. Both resting cells and crude enzyme of Rhodococcus sp. UKMP-5M were able to biodegrade cyanide to ammonia and formate without the formation of formamide, implying the identification of a simple hydrolytic cyanide degradation pathway involving the enzyme cyanidase. Further verification with SDS-PAGE revealed that the molecular weight of the active enzyme was estimated to be 38 kDa, which is consistent with previously reported cyanidases. Since the recent advancement in the application of biological methods in treating cyanide-bearing wastewater has been promising, the discovery of this new bacterium will add value by diversifying the existing microbial populations capable of cyanide detoxification.

  14. Biodegradation of variable-chain-length alkanes at low temperatures by a psychrotrophic Rhodococcus sp.

    SciTech Connect

    Whyte, L.G.; Hawari, J.; Zhou, E.; Bourbonniere, L.; Greer, C.W.; Inniss, W.E.

    1998-07-01

    The psychrotroph Rhodococcus sp. strain Q15 was examined for its ability to degrade individual n-alkanes and diesel fuel at low temperatures, and its alkane catabolic pathway was investigated by biochemical and genetic techniques. At 0 and 5 C, Q15 mineralized the short-chain alkanes dodecane and hexadecane to a greater extent than that observed for the long-chain alkanes octacosane and dotriacontane. Q15 utilized a broad range of aliphatics (C{sub 10} to C{sub 21} alkanes, branched alkanes, and a substituted cyclohexane) present in diesel fuel at 5 C. Mineralization of hexadecane at 5 C was significantly greater in both hydrocarbon-contaminated and pristine soil microcosms seeded with Q15 cells than in uninoculated control soil microcosms. The detection of hexadecane and dodecane metabolic intermediates (1-hexadecanol and 2-hexadecanol and 1-do-decanol and 2-dodecanone, respectively) by solid-phase microextraction-gas chromatography-mass spectrometry and the utilization of potential metabolic intermediates indicated that Q15 oxidizes alkanes by both the terminal oxidation pathway and the subterminal oxidation pathway. Genetic characterization by PCR and nucleotide sequence analysis indicated that Q15 possesses an aliphatic aldehyde dehydrogenase gene highly homologous to the Rhodococcus erythropolis thcA gene. Rhodococcus sp. strain Q15 possessed two large plasmids of approximately 90 and 115 kb (shown to mediate Cd resistance) which were not required for alkane mineralization, although the 90-kb plasmid enhanced mineralization of some alkanes and growth on diesel oil at both 5 and 25 C.

  15. Biodegradation of Variable-Chain-Length Alkanes at Low Temperatures by a Psychrotrophic Rhodococcus sp.

    PubMed Central

    Whyte, Lyle G.; Hawari, Jalal; Zhou, Edward; Bourbonnière, Luc; Inniss, William E.; Greer, Charles W.

    1998-01-01

    The psychrotroph Rhodococcus sp. strain Q15 was examined for its ability to degrade individual n-alkanes and diesel fuel at low temperatures, and its alkane catabolic pathway was investigated by biochemical and genetic techniques. At 0 and 5°C, Q15 mineralized the short-chain alkanes dodecane and hexadecane to a greater extent than that observed for the long-chain alkanes octacosane and dotriacontane. Q15 utilized a broad range of aliphatics (C10 to C21 alkanes, branched alkanes, and a substituted cyclohexane) present in diesel fuel at 5°C. Mineralization of hexadecane at 5°C was significantly greater in both hydrocarbon-contaminated and pristine soil microcosms seeded with Q15 cells than in uninoculated control soil microcosms. The detection of hexadecane and dodecane metabolic intermediates (1-hexadecanol and 2-hexadecanol and 1-dodecanol and 2-dodecanone, respectively) by solid-phase microextraction–gas chromatography-mass spectrometry and the utilization of potential metabolic intermediates indicated that Q15 oxidizes alkanes by both the terminal oxidation pathway and the subterminal oxidation pathway. Genetic characterization by PCR and nucleotide sequence analysis indicated that Q15 possesses an aliphatic aldehyde dehydrogenase gene highly homologous to the Rhodococcus erythropolis thcA gene. Rhodococcus sp. strain Q15 possessed two large plasmids of approximately 90 and 115 kb (shown to mediate Cd resistance) which were not required for alkane mineralization, although the 90-kb plasmid enhanced mineralization of some alkanes and growth on diesel oil at both 5 and 25°C. PMID:9647833

  16. A new zearalenone biodegradation strategy using non-pathogenic Rhodococcus pyridinivorans K408 strain.

    PubMed

    Kriszt, Rókus; Krifaton, Csilla; Szoboszlay, Sándor; Cserháti, Mátyás; Kriszt, Balázs; Kukolya, József; Czéh, Arpád; Fehér-Tóth, Szilvia; Török, Lívia; Szőke, Zsuzsanna; Kovács, Krisztina J; Barna, Teréz; Ferenczi, Szilamér

    2012-01-01

    Zearalenone (hereafter referred to as ZEA) is a nonsteroidal estrogenic mycotoxin produced by several Fusarium spp. on cereal grains. ZEA is one of the most hazardous natural endocrine disrupting chemicals (EDC) which induces hyper estrogenic responses in mammals. This can result in reproductive disorders in farm animals as well as in humans. Consequently, detoxification strategies for contaminated crops are crucial for food safety. In this study we have developed a bacterial based detoxification system using a non-pathogen Rhodococcus pyridinivorans K408 strain. Following 5 days treatment of ZEA with R. pyridinivorans K408 strain HPLC analyses showed an 87.21% ZEA-degradation efficiency of the bacterial enzyme systems. In another approach, the strain biotransformation ability has also been confirmed by a bioluminescent version of the yeast estrogen screening system (BLYES), which detected an 81.75% of biodegradability of ZEA, in a good agreement with the chemical analyses. Furthermore, the capacity of R. pyridinivorans to eliminate the estrogenic effects of ZEA was tested by using an immature uterotrophic assay. Prepubertal female rats were treated with vehicle (olive oil), 17β-estradiol, ZEA (0.1-1-5-10 mg/kg body weight) and LB broth containing 500 mg/l ZEA that has already been incubated with or without Rhodococcus pyridinivorans K408 strain. Uterine weights were measured and the mRNA level changes relating to apelin, aquaporin 5, complement component 2, and calbindin-3 genes were measured by qRT-PCR. These genes represent the major pathways that are affected by estromimetic compounds. Zearalenone feeding significantly increased the uterus weight in a dose dependent manner and at the same time upregulated complement component 2 and calbindin-3 expression as well as decreased apelin and aquaporin 5 mRNA levels comparable to that seen in 17β-estradiol exposed rats. In contrast, LB broth in which ZEA was incubated with Rhodococcus pyridinivorans K408 prior to

  17. Rhodococcus equi Sepsis in a Renal Transplant Recipient: A Case Study

    PubMed Central

    Macken, Eline; de Jonge, Hylke; Van Caesbroeck, Daniël; Verhaegen, Jan; Van Kerkhoven, Dana; Van Wijngaerden, Eric; Kuypers, Dirk

    2015-01-01

    Abstract Rhodococcus equi is an unusual cause of infection in humans, but has emerged as an opportunistic pathogen among immunocompromised patients. Primary pulmonary involvement is the most common clinical presentation, although the spectrum of disease is broad. Diagnosing R. equi infections remains challenging, both from clinical and microbiological view, and no standard treatment has been established. In this report, we present a detailed case of a 57-year-old male renal transplant recipient who developed R. equi bacteremia with a concomitant Pneumocystis jirovecii pneumonia. We describe the clinical features of R. equi infections, highlight the importance of an early diagnosis, and briefly review treatment options for this rare infection. PMID:27500216

  18. Activity of clarithromycin or rifampin alone or in combination against experimental Rhodococcus equi infection in mice.

    PubMed

    Burton, Alexandra J; Giguère, Steeve; Berghaus, Londa J; Hondalus, Mary K

    2015-01-01

    Treatment of mice with the combination of clarithromycin with rifampin resulted in a significantly lower number of Rhodococcus equi CFU in the organs of mice than treatment with either drug alone or placebo. There was no significant difference in the number of R. equi CFU between mice treated with clarithromycin monotherapy, rifampin monotherapy, or placebo. The combination of clarithromycin with rifampin conferred a clear advantage over either drug as monotherapy in this model of chronic R. equi infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  19. Regio- and stereoselective oxidation of unactivated C–H bonds with Rhodococcus rhodochrous

    PubMed Central

    O’Reilly, Elaine; Aitken, Suzanne J; Grogan, Gideon; Kelly, Paul P; Turner, Nicholas J

    2012-01-01

    Summary The ability of Rhodococcus rhodochrous (NCIMB 9703) to catalyse the regio- and stereoselective hydroxylation of a range of benzyloxy-substituted heterocycles has been investigated. Incubation of 2-benzyloxytetrahydropyrans with resting cell suspensions of the organism yielded predominantly a mixture of 5-hydroxylated isomers in combined yields of up to 40%. Exposure of the corresponding 2-benzyloxytetrahydrofuran derivatives to the cell suspensions gave predominantly the 4-hydroxylated isomers in yields of up to 26%. Most interestingly, 2-(4-nitrobenzyloxy)tetrahydrofuran and 2-(4-nitrobenzyloxy)tetrahydropyran were transformed in high yields to the 4-hydroxylated and 5-hydroxylated products, respectively. PMID:22509221

  20. Cloning and sequence analysis of the gene encoding isocitrate lyase from Rhodococcus fascians.

    PubMed

    Vereecke, D; Villarroel, R; Van Montagu, M; Desomer, J

    1994-07-22

    An isocitrate lyase (Icl)-encoding gene (icl) from the Gram+ plant pathogen Rhodococcus fascians was identified serendipitously as part of a scrambled fragment after shotgun cloning in the promoter probe vector, pDP1. The Icl protein is 429 amino acids long (47.11 kDa) and has a predicted pI of 4.84; it is 54% similar to the Escherichia coli Icl and 24-27% to eukaryotic homologues. Comparison of the prokaryotic and eukaryotic Icl confirms the earlier proposal of Matsuoka and McFadden [J. Bacteriol. 143 (1988) 4528-4536] that the enzyme has enlarged during evolution.

  1. Phenotypic alterations in Arabidopsis thaliana plants caused by Rhodococcus fascians infection.

    PubMed

    de O Manes, Carmem-Lara; Beeckman, Tom; Ritsema, Tita; Van Montagu, Marc; Goethals, Koen; Holsters, Marcelle

    2004-04-01

    Arabidopsis thaliana (L.) Heynh. plants were challenged with Rhodococcus fascians at several developmental stages and using different inoculation procedures. A variety of morphological alterations was scored on the infected plants; some of them resembled phenotypes of A. thaliana mutants in their shoot apical meristem (SAM) organization. Infection with R. fascians did not affect SAM organization in wild type nor in SAM mutants. Anatomical studies on the new organs formed after infection with R. fascians demonstrated extensive bacterial colonization. Colonization and concomitant production of specific signals are the likely cause of malformations.

  2. The fas locus of the phytopathogen Rhodococcus fascians affects mitosis of tobacco BY-2 cells.

    PubMed

    Temmerman, W; Ritsema, T; Simón-Mateo, C; Van Montagu, M; Mironov, V; Inzé, D; Goethals, K; Holsters, M

    2001-03-09

    The effect of Rhodococcus fascians, the causal agent of leafy gall disease, on the mitotic behavior of synchronized tobacco Bright Yellow-2 (BY-2) cells was investigated. Incubation of aphidicolin-synchronized BY-2 cells with R. fascians cells specifically resulted in a broader mitotic index peak, an effect that was linked to an intact and expressed fas virulence locus. The obtained results pointed towards an effect of R. fascians on the prophase of mitosis. The relevance of these results to the virulence of the bacterium is discussed.

  3. Novel Allylic Oxidation of α-Cedrene to sec-Cedrenol by a Rhodococcus Strain

    PubMed Central

    Takigawa, Hirofumi; Kubota, Hiromi; Sonohara, Hiroshi; Okuda, Mitsuyoshi; Tanaka, Shigeyoshi; Fujikura, Yoshiaki; Ito, Susumu

    1993-01-01

    A bacterial strain, designated KSM-7358, that can use α-cedrene for growth was isolated. The strain was identified as a member of the genus Rhodococcus and catalyzed the novel allylic oxidation of α-cedrene regiospecifically to produce (R)-10-hydroxycedrene (sec-cedrenol) with a very high yield. α-Curcumene was also produced as a possible metabolite of sec-cedrenol. A possible pathway for the microbial conversion of α-cedrene to sec-cedrenol and α-curcumene is proposed. PMID:16348930

  4. Identification of a heroin esterase in Rhodococcus sp. strain H1.

    PubMed Central

    Cameron, G W; Jordan, K N; Holt, P J; Baker, P B; Lowe, C R; Bruce, N C

    1994-01-01

    A strain of a Rhodococcus sp. (termed H1) capable of utilizing heroin as its sole carbon and energy source was isolated by selective enrichment. An inducible heroin esterase was partially purified and shown to catalyze the hydrolysis of both of the acetylester groups of heroin. The enzyme displays optimum activity at pH 8.5 and appears to be a trimer of identical subunits with an M(r) or 39,000 and a native M(r) of 120,000. PMID:7986057

  5. A New Zearalenone Biodegradation Strategy Using Non-Pathogenic Rhodococcus pyridinivorans K408 Strain

    PubMed Central

    Kriszt, Rókus; Krifaton, Csilla; Szoboszlay, Sándor; Cserháti, Mátyás; Kriszt, Balázs; Kukolya, József; Czéh, Árpád; Fehér-Tóth, Szilvia; Török, Lívia; Szőke, Zsuzsanna; Kovács, Krisztina J.; Barna, Teréz; Ferenczi, Szilamér

    2012-01-01

    Zearalenone (hereafter referred to as ZEA) is a nonsteroidal estrogenic mycotoxin produced by several Fusarium spp. on cereal grains. ZEA is one of the most hazardous natural endocrine disrupting chemicals (EDC) which induces hyper estrogenic responses in mammals. This can result in reproductive disorders in farm animals as well as in humans. Consequently, detoxification strategies for contaminated crops are crucial for food safety. In this study we have developed a bacterial based detoxification system using a non-pathogen Rhodococcus pyridinivorans K408 strain. Following 5 days treatment of ZEA with R. pyridinivorans K408 strain HPLC analyses showed an 87.21% ZEA-degradation efficiency of the bacterial enzyme systems. In another approach, the strain biotransformation ability has also been confirmed by a bioluminescent version of the yeast estrogen screening system (BLYES), which detected an 81.75% of biodegradability of ZEA, in a good agreement with the chemical analyses. Furthermore, the capacity of R. pyridinivorans to eliminate the estrogenic effects of ZEA was tested by using an immature uterotrophic assay. Prepubertal female rats were treated with vehicle (olive oil), 17β-estradiol, ZEA (0.1-1-5-10 mg/kg body weight) and LB broth containing 500 mg/l ZEA that has already been incubated with or without Rhodococcus pyridinivorans K408 strain. Uterine weights were measured and the mRNA level changes relating to apelin, aquaporin 5, complement component 2, and calbindin-3 genes were measured by qRT-PCR. These genes represent the major pathways that are affected by estromimetic compounds. Zearalenone feeding significantly increased the uterus weight in a dose dependent manner and at the same time upregulated complement component 2 and calbindin-3 expression as well as decreased apelin and aquaporin 5 mRNA levels comparable to that seen in 17β-estradiol exposed rats. In contrast, LB broth in which ZEA was incubated with Rhodococcus pyridinivorans K408 prior to

  6. Cyanobactericidal effect of Rhodococcus sp. isolated from eutrophic lake on Microcystis sp.

    PubMed

    Lee, Young-Ki; Ahn, Chi-Yong; Kim, Hee-Sik; Oh, Hee-Mock

    2010-11-01

    A bacterium, which was observed in all cultivations of Microcystis sp., was isolated and designated as Rhodococcus sp. KWR2. The growth of bloom-forming cyanobacteria, including four strains of Microcystis aeruginosa and Anabaena variabilis, was suppressed by up to 75-88% by 2% (v/v) culture broth of KWR2 after 5 days. But KWR2 did not inhibit eukaryotic algae, Chlorella vulgaris and Scenedesmus sp. An extracellular algicidal substance produced by KWR2 showed a cyanobactericidal activity of 94% and was water-soluble with a molecular weight of lower than 8 kDa.

  7. Statistical optimization for production of chitin deacetylase from Rhodococcus erythropolis HG05.

    PubMed

    Sun, Yuying; Zhang, Jiquan; Wu, Shengjun; Wang, Shujun

    2014-02-15

    A strain producing chitin deacetylase (CDA) was isolated and identified as Rhodococcus erythropolis by morphological characteristics and 16S rDNA analysis, named as R. erythropolis HG05. By Plackett-Burman and central composite design, CDA production from R. erythropolis HG05 was increased from 58.00 U/mL to 238.89 U/mL. With the crude enzyme from R. erythropolis HG05, the hydrolysate components from colloid chitin were chito-oligosaccharides with polymerization number larger than hexaose.

  8. A Leaf-Inhabiting Endophytic Bacterium, Rhodococcus sp. KB6, Enhances Sweet Potato Resistance to Black Rot Disease Caused by Ceratocystis fimbriata.

    PubMed

    Hong, Chi Eun; Jeong, Haeyoung; Jo, Sung Hee; Jeong, Jae Cheol; Kwon, Suk Yoon; An, Donghwan; Park, Jeong Mee

    2016-03-01

    Rhodococcus species have become increasingly important owing to their ability to degrade a wide range of toxic chemicals and produce bioactive compounds. Here, we report isolation of the Rhodococcus sp. KB6, which is a new leaf-inhabiting endophytic bacterium that suppresses black rot disease in sweet potato leaves. We determined the 7.0 Mb draft genome sequence of KB6 and have predicted 19 biosynthetic gene clusters for secondary metabolites, including heterobactins, which are a new class of siderophores. Notably, we showed the first internal colonization of host plants with Rhodococcus sp. KB6 and discuss its potential as a biocontrol agent for sustainable agriculture.

  9. Structural insights into substrate specificity and solvent tolerance in alcohol dehydrogenase ADH-'A' from Rhodococcus ruber DSM 44541.

    PubMed

    Karabec, Martin; Łyskowski, Andrzej; Tauber, Katharina C; Steinkellner, Georg; Kroutil, Wolfgang; Grogan, Gideon; Gruber, Karl

    2010-09-14

    The structure of the alcohol dehydrogenase ADH-'A' from Rhodococcus ruber reveals possible reasons for its remarkable tolerance to organic co-solvents and suggests new directions for structure-informed mutagenesis to produce enzymes of altered substrate specificity or improved selectivity.

  10. The Explosive-Degrading Cytochrome P450 System Is Highly Conserved among Strains of Rhodococcus spp.▿

    PubMed Central

    Seth-Smith, Helena M. B.; Edwards, James; Rosser, Susan J.; Rathbone, Deborah A.; Bruce, Neil C.

    2008-01-01

    Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a widely used explosive and a serious environmental pollutant. Nineteen strains of Rhodococcus spp. capable of utilizing RDX as the sole nitrogen source have been isolated. The cytochrome P450 system XplA-XplB, which is responsible for RDX breakdown, is present in 18 of these strains. PMID:18487400

  11. Complete Genome of Rhodococcus pyridinivorans SB3094, a Methyl-Ethyl-Ketone-Degrading Bacterium Used for Bioaugmentation

    PubMed Central

    Albertsen, Mads; D’Imperio, Seth; Tale, Vaibhav P.; Lewis, Derrick; Nielsen, Per Halkjær; Nielsen, Jeppe Lund

    2014-01-01

    Here, we present the complete genome of Rhodococcus pyridinivorans SB3094, a methyl-ethyl-ketone (MEK)-degrading strain used for bioaugmentation relating to the treatment of wastewater contamination with petrochemical hydrocarbons. The genome highlights important features for bioaugmentation, including the genes involved in the degradation of MEK. PMID:24874690

  12. Complete Genome of Rhodococcus pyridinivorans SB3094, a Methyl-Ethyl-Ketone-Degrading Bacterium Used for Bioaugmentation.

    PubMed

    Dueholm, Morten S; Albertsen, Mads; D'Imperio, Seth; Tale, Vaibhav P; Lewis, Derrick; Nielsen, Per Halkjær; Nielsen, Jeppe Lund

    2014-05-29

    Here, we present the complete genome of Rhodococcus pyridinivorans SB3094, a methyl-ethyl-ketone (MEK)-degrading strain used for bioaugmentation relating to the treatment of wastewater contamination with petrochemical hydrocarbons. The genome highlights important features for bioaugmentation, including the genes involved in the degradation of MEK.

  13. Insight on RDX degradation mechanism by Rhodococcus strains using 13C and 15N kinetic isotope effects.

    PubMed

    Bernstein, Anat; Ronen, Zeev; Gelman, Faina

    2013-01-02

    The explosive Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is known to be degraded aerobically by various isolates of the Rhodococcus species, with denitration being the key step, mediated by Cytochrome P450. Our study aimed at gaining insight into the RDX degradation mechanism by Rhodococcus species and comparing isotope effects associated with RDX degradation by distinct Rhodococcus strains. For these purposes, enrichment in (13)C and (15)N isotopes throughout RDX denitration was studied for three distinct Rhodococcus strains, isolated from soil and groundwater in an RDX-contaminated site. The observable (15)N enrichment throughout the reaction, together with minor (13)C enrichment, suggests that N-N bond cleavage is likely to be the key rate-limiting step in the reaction. The similarity in the kinetic (15)N isotope effect between the three tested strains suggests that either isotope-masking effects are negligible, or are of a similar extent for all tested strains. The lack of variability in the kinetic (15)N isotope effect allows the interpretation of environmental studies with greater confidence.

  14. Draft Genome Sequence of Rhodococcus erythropolis NSX2, an Actinobacterium Isolated from a Cadmium-Contaminated Environment.

    PubMed

    Egidi, Eleonora; Wood, Jennifer L; Fox, Edward M; Liu, Wuxing; Franks, Ashley E

    2016-10-20

    Rhodococcus erythropolis NSX2 is a rhizobacterium isolated from a heavy metal-contaminated environment. The 6.2-Mb annotated genome sequence shows that this strain harbors genes associated with heavy-metal resistance and xenobiotics degradation. Copyright © 2016 Egidi et al.

  15. Transfer of the virulence-associated protein A-bearing plasmid between field strains of virulent and avirulent Rhodococcus equi

    USDA-ARS?s Scientific Manuscript database

    Virulent and avirulent isolates coexist in equine feces and the environment and serve as a source of infection for foals. The extent to which conjugative plasmid transfer occurs between these strains is unknown and is important for understanding the epidemiology of Rhodococcus equi infections of fo...

  16. Sequence and molecular characterization of a DNA region encoding the dibenzothiophene desulfurization operon of Rhodococcus sp. strain IGTS8.

    PubMed

    Piddington, C S; Kovacevich, B R; Rambosek, J

    1995-02-01

    Dibenzothiophene (DBT), a model compound for sulfur-containing organic molecules found in fossil fuels, can be desulfurized to 2-hydroxybiphenyl (2-HBP) by Rhodococcus sp. strain IGTS8. Complementation of a desulfurization (dsz) mutant provided the genes from Rhodococcus sp. strain IGTS8 responsible for desulfurization. A 6.7-kb TaqI fragment cloned in Escherichia coli-Rhodococcus shuttle vector pRR-6 was found to both complement this mutation and confer desulfurization to Rhodococcus fascians, which normally is not able to desulfurize DBT. Expression of this fragment in E. coli also conferred the ability to desulfurize DBT. A molecular analysis of the cloned fragment revealed a single operon containing three open reading frames involved in the conversion of DBT to 2-HBP. The three genes were designated dszA, dszB, and dszC. Neither the nucleotide sequences nor the deduced amino acid sequences of the enzymes exhibited significant similarity to sequences obtained from the GenBank, EMBL, and Swiss-Prot databases, indicating that these enzymes are novel enzymes. Subclone analyses revealed that the gene product of dszC converts DBT directly to DBT-sulfone and that the gene products of dszA and dszB act in concert to convert DBT-sulfone to 2-HBP.

  17. Metagenome Sequencing Reveals Rhodococcus Dominance in Farpuk Cave, Mizoram, India, an Eastern Himalayan Biodiversity Hot Spot Region

    PubMed Central

    De Mandal, Surajit; Sanga, Zothan

    2015-01-01

    The present study employed 16S rRNA amplicon sequencing to survey the prokaryotic microbiota on Farpuk Cave, revealing a diverse bacterial community with 4,021 operational taxonomical units (OTUs), mainly dominated by the genus Rhodococcus. Moreover, 18.17% of the OTUs were unclassified at the phylum level, suggesting the existence of novel bacterial species. PMID:26067958

  18. [Cloning and analysis of a new aliphatic amidase gene from Rhodococcus erythropolis TA37].

    PubMed

    Lavrov, K V; Karpova, I Yu; Epremyan, A S; Yanenko, A S

    2014-10-01

    A new aliphatic amidase gene (ami), having a level of similarity with the nearest homologs of no more than 77%, was identified in the Rhodococcus erythropolis TA37 strain, which is able to hydrolyze a wide range of amides. The amidase gene was cloned within a 3.7 kb chromosomal locus, which also contains putative acetyl-CoA ligase and ABC-type transportergenes. The structure of this locus in the R. erythropolis TA37 strain differs from the structure of loci in other Rhodococcus strains. The amidase gene is expressed in Escherichia coli cells. It was demonstrated that amidase (generated in the recombinant strain) efficiently hydrolyzes acetamide (aliphatic anmide) and does not use 4'-nitroacetanilide (N-substituted amide) as a substrate. Insertional inactivation of the amidase gene in the R. erythropolis TA37 strain results in a considerable decrease (by at least 6-7 times) in basal amidase activity, indicating functional amidase activity in the R. erythropolis TA37 strain.

  19. An Extremely Oligotrophic Bacterium, Rhodococcus erythropolis N9T-4, Isolated from Crude Oil▿

    PubMed Central

    Ohhata, Naoko; Yoshida, Nobuyuki; Egami, Hiroshi; Katsuragi, Tohoru; Tani, Yoshiki; Takagi, Hiroshi

    2007-01-01

    Rhodococcus erythropolis N9T-4, which was isolated from crude oil, showed extremely oligotrophic growth and formed its colonies on a minimal salt medium solidified using agar or silica gel without any additional carbon source. N9T-4 did not grow under CO2-limiting conditions but could grow on a medium containing NaHCO3 under the same conditions, suggesting that the oligotrophic growth of N9T-4 depends on CO2. Proteomic analysis of N9T-4 revealed that two proteins, with molecular masses of 45 and 55 kDa, were highly induced under the oligotrophic conditions. The primary structures of these proteins exhibited striking similarities to those of methanol: N,N′-dimethyl-4-nitrosoaniline oxidoreductase and an aldehyde dehydrogenase from Rhodococcus sp. These enzyme activities were three times higher under oligotrophic conditions than under n-tetradecane-containing heterotrophic conditions, and gene disruption for the aldehyde dehydrogenase caused a lack of growth on the minimal salt medium. Furthermore, 3-hexulose 6-phosphate synthase and phospho-3-hexuloisomerase activities, which are key enzymes in the ribulose monophosphate pathway in methylotrophic bacteria, were detected specifically in the cell extract of oligotrophically grown N9T-4. These results suggest that CO2 fixation involves methanol (formaldehyde) metabolism in the oligotrophic growth of R. erythropolis N9T-4. PMID:17675378

  20. Identification and Characterization of a Tetramethylpyrazine Catabolic Pathway in Rhodococcus jostii TMP1

    PubMed Central

    Stankeviciute, Jonita; Urbelis, Gintaras; Tauraite, Daiva; Rutkiene, Rasa; Meskys, Rolandas

    2013-01-01

    At present, there are no published data on catabolic pathways of N-heterocyclic compounds, in which all carbon atoms carry a substituent. We identified the genetic locus and characterized key reactions in the aerobic degradation of tetramethylpyrazine in Rhodococcus jostii strain TMP1. By comparing protein expression profiles, we identified a tetramethylpyrazine-inducible protein of 40 kDa and determined its identity by tandem mass spectrometry (MS-MS) de novo sequencing. Searches against an R. jostii TMP1 genome database allowed the identification of the tetramethylpyrazine-inducible protein-coding gene. The tetramethylpyrazine-inducible gene was located within a 13-kb genome cluster, denominated the tetramethylpyrazine degradation (tpd) locus, that encoded eight proteins involved in tetramethylpyrazine catabolism. The genes from this cluster were cloned and transferred into tetramethylpyrazine-nondegrading Rhodococcus erythropolis strain SQ1. This allowed us to verify the function of the tpd locus, to isolate intermediate metabolites, and to reconstruct the catabolic pathway of tetramethylpyrazine. We report that the degradation of tetramethylpyrazine is a multistep process that includes initial oxidative aromatic-ring cleavage by tetramethylpyrazine oxygenase, TpdAB; subsequent hydrolysis by (Z)-N,N′-(but-2-ene-2,3-diyl)diacetamide hydrolase, TpdC; and further intermediate metabolite reduction by aminoalcohol dehydrogenase, TpdE. Thus, the genes responsible for bacterial degradation of pyrazines have been identified, and intermediate metabolites of tetramethylpyrazine degradation have been isolated for the first time. PMID:23563941

  1. Genome and proteome analysis of phage E3 infecting the soil-borne actinomycete Rhodococcus equi.

    PubMed

    Salifu, Samson P; Valero-Rello, Ana; Campbell, Samantha A; Inglis, Neil F; Scortti, Mariela; Foley, Sophie; Vázquez-Boland, José A

    2013-02-01

    We report on the characterization and genomic analysis of bacteriophage E3 isolated from soil and propagating in Rhodococcus equi strains. Phage E3 has a circular genome of 142 563 bp and is the first Myoviridae reported for the genus Rhodococcus and for a non-mycobacterial actinomycete. Phylogenetic analyses placed E3 in a distinct Myoviridae clade together with Mycobacterium phages Bxz1 and Myrna. The highly syntenic genomes of this myoviridal group comprise vertically evolving core phage modules flanked by hyperplastic regions specific to each phage and rich in horizontally acquired DNA. The hyperplastic regions contain numerous tRNA genes in the mycobacteriophages which are absent in E3, possibly reflecting bacterial host-specific translation-related phage fitness constraints associated with rate-limiting tRNAs. A structural proteome analysis identified 28 E3 polypeptides, including 15 not previously known to be virion-associated proteins. The E3 genome and comparative analysis provide insight into short-term genome evolution and adaptive plasticity in tailed phages from the environmental microbiome. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

  2. Conjugal Transfer of a Virulence Plasmid in the Opportunistic Intracellular Actinomycete Rhodococcus equi

    PubMed Central

    Tripathi, V. N.; Harding, W. C.; Willingham-Lane, J. M.

    2012-01-01

    Rhodococcus equi is a facultative intracellular, Gram-positive, soilborne actinomycete which can cause severe pyogranulomatous pneumonia with abscessation in young horses (foals) and in immunocompromised people, such as persons with AIDS. All strains of R. equi isolated from foals and approximately a third isolated from humans contain a large, ∼81-kb plasmid which is essential for the intramacrophage growth of the organism and for virulence in foals and murine in vivo model systems. We found that the entire virulence plasmid could be transferred from plasmid-containing strains of R. equi (donor) to plasmid-free R. equi strains (recipient) at a high frequency and that plasmid transmission reestablished the capacity for intracellular growth in macrophages. Plasmid transfer required living cells and cell-to-cell contact and was unaffected by the presence of DNase, factors pointing to conjugation as the major means of genetic transfer. Deletion of a putative relaxase-encoding gene, traA, located in the proposed conjugative region of the plasmid, abolished plasmid transfer. Reversion of the traA mutation restored plasmid transmissibility. Finally, plasmid transmission to other Rhodococcus species and some additional related organisms was demonstrated. This is the first study showing a virulence plasmid transfer in R. equi, and it establishes a mechanism by which the virulence plasmid can move among bacteria in the soil. PMID:23042997

  3. Conjugal transfer of a virulence plasmid in the opportunistic intracellular actinomycete Rhodococcus equi.

    PubMed

    Tripathi, V N; Harding, W C; Willingham-Lane, J M; Hondalus, M K

    2012-12-01

    Rhodococcus equi is a facultative intracellular, Gram-positive, soilborne actinomycete which can cause severe pyogranulomatous pneumonia with abscessation in young horses (foals) and in immunocompromised people, such as persons with AIDS. All strains of R. equi isolated from foals and approximately a third isolated from humans contain a large, ~81-kb plasmid which is essential for the intramacrophage growth of the organism and for virulence in foals and murine in vivo model systems. We found that the entire virulence plasmid could be transferred from plasmid-containing strains of R. equi (donor) to plasmid-free R. equi strains (recipient) at a high frequency and that plasmid transmission reestablished the capacity for intracellular growth in macrophages. Plasmid transfer required living cells and cell-to-cell contact and was unaffected by the presence of DNase, factors pointing to conjugation as the major means of genetic transfer. Deletion of a putative relaxase-encoding gene, traA, located in the proposed conjugative region of the plasmid, abolished plasmid transfer. Reversion of the traA mutation restored plasmid transmissibility. Finally, plasmid transmission to other Rhodococcus species and some additional related organisms was demonstrated. This is the first study showing a virulence plasmid transfer in R. equi, and it establishes a mechanism by which the virulence plasmid can move among bacteria in the soil.

  4. Characterization of four Rhodococcus alcohol dehydrogenase genes responsible for the oxidation of aromatic alcohols.

    PubMed

    Peng, Xue; Taki, Hironori; Komukai, Syoko; Sekine, Mitsuo; Kanoh, Kaneo; Kasai, Hiroaki; Choi, Seon-Kang; Omata, Seiha; Tanikawa, Satoshi; Harayama, Shigeaki; Misawa, Norihiko

    2006-08-01

    Four genes were isolated and characterized for alcohol dehydrogenases (ADHs) catalyzing the oxidation of aromatic alcohols such as benzyl alcohol to their corresponding aldehydes, one from o-xylene-degrading Rhodococcus opacus TKN14 and the other three from n-alkane-degrading Rhodococcus erythropolis PR4. Various aromatic alcohols were bioconverted to their corresponding carboxylic acids using Escherichia coli cells expressing each of the four ADH genes together with an aromatic aldehyde dehydrogenase gene (phnN) from Sphingomonas sp. strain 14DN61. The ADH gene (designated adhA) from strain TKN14 had the ability to biotransform a wide variety of aromatic alcohols, i.e., 2-hydroxymethyl-6-methylnaphthalene, 2-hydroxymethylnaphthalene, xylene-alpha,alpha'-diol, 3-chlorobenzyl alcohol, and vanillyl alcohol, in addition to benzyl alcohol with or without a hydroxyl, methyl, or methoxy substitution. In contrast, the three ADH genes of strain PR4 (designated adhA, adhB, and adhC) exhibited lower ability to degrade these alcohols: these genes stimulated the conversion of the alcohol substrates by only threefold or less of the control value. One exception was the conversion of 3-methoxybenzyl alcohol, which was stimulated sevenfold by adhB. A phylogenetic analysis of the amino acid sequences of these four enzymes indicated that they differed from other Zn-dependent ADHs.

  5. Influence of Rhodococcus equi on the respiratory burst of resident alveolar macrophages from horses

    SciTech Connect

    Brumbaugh, G.W.

    1986-01-01

    Rhodococcus equi is the etiologic agent of a devastating pneumonia of sporadic incidence in foals. The purpose of this study was to evaluate the influence of R. equi on the superoxide anion production, measured spectrophotometrically as the reduction of cytochrome C, and hexose monophosphate shunt activity, measured by /sup 14/CO/sub 2/ liberation from /sup 14/C-1-D-glucose, of alveolar macrophages from horses. Alveolar macrophages were harvested from 6 anesthetized, healthy, light-breed, adult horses by bronchoalveolar lavage. Following a randomized complete block design, the suspension of cells was divided into aliquots of 10/sup 6/ viable alveolar macrophages which were exposed to 1, 10 or 100 g. of opsonized R. equi or opsonized zymosan A at 37 C for 2 hours. In this study the respiratory burst of equine alveolar macrophages was only evidenced by the hexose monophosphate shunt activity and superoxide anion was not coincidentally produced. Rhodococcus equi did not adversely affect that response. The insignificant superoxide anion production by the alveolar macrophages suggests that this may not be a significant oxygen metabolite in those cells.

  6. Expression, purification and kinetic characterization of recombinant benzoate dioxygenase from Rhodococcus ruber UKMP-5M

    PubMed Central

    Tavakoli, Arezoo; Hamzah, Ainon; Rabu, Amir

    2016-01-01

    In this study, benzoate dioxygenase from Rhodococcus ruber UKMP-5M was catalyzed by oxidating the benzene ring to catechol and other derivatives. The benzoate dioxygenase (benA gene) from Rhodococcus ruber UKMP-5M was then expressed, purified, characterized, The benA gene was amplified (642 bp), and the product was cloned into a pGEM-T vector. The recombinant plasmid pGEMT-benA was digested by double restriction enzymes BamHI and HindIII to construct plasmid pET28b-benA and was then ligated into Escherichia coli BL21 (DE3). The recombinant E. coli was induced with 0.5 mM isopropyl β-D-thiogalactoside (IPTG) at 22˚C to produce benzoate dioxygenase. The enzyme was then purified by ion exchange chromatography after 8 purification folds. The resulting product was 25 kDa, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Benzoate dioxygenase activity was found to be 6.54 U/mL and the optimal pH and temperature were 8.5 and 25°C, respectively. Maximum velocity (Vmax) and Michaelis constant (Km) were 7.36 U/mL and 5.58 µM, respectively. The end metabolite from the benzoate dioxygenase reaction was cyclohexane dione, which was determined by gas chromatography mass spectrometry (GC-MS). PMID:28097167

  7. Biosurfactant production by halotolerant Rhodococcus fascians from Casey Station, Wilkes Land, Antarctica.

    PubMed

    Gesheva, Victoria; Stackebrandt, Erko; Vasileva-Tonkova, Evgenia

    2010-08-01

    Isolate A-3 from Antarctic soil in Casey Station, Wilkes Land, was characterized for growth on hydrocarbons. Use of glucose or kerosene as a sole carbon source in the culture medium favoured biosynthesis of surfactant which, by thin-layer chromatography, indicated the formation of a rhamnose-containing glycolipid. This compound lowered the surface tension at the air/water interface to 27 mN/m as well as inhibited the growth of B. subtilis ATCC 6633 and exhibited hemolytic activity. A highly hydrophobic surface of the cells suggests that uptake occurs via a direct cell-hydrocarbon substrate contact. Strain A-3 is Gram-positive, halotolerant, catalase positive, urease negative and has rod-coccus shape. Its cell walls contained meso-diaminopimelic acid. Phylogenetic analysis based on comparative analysis of 16S rRNA gene sequences revealed that strain A-3 is closely related to Rhodococcus fascians with which it shares 100% sequence similarity. This is the first report on rhamnose-containing biosurfactant production by Rhodococcus fascians isolated from Antarctic soil.

  8. Identification and characterization of a tetramethylpyrazine catabolic pathway in Rhodococcus jostii TMP1.

    PubMed

    Kutanovas, Simonas; Stankeviciute, Jonita; Urbelis, Gintaras; Tauraite, Daiva; Rutkiene, Rasa; Meskys, Rolandas

    2013-06-01

    At present, there are no published data on catabolic pathways of N-heterocyclic compounds, in which all carbon atoms carry a substituent. We identified the genetic locus and characterized key reactions in the aerobic degradation of tetramethylpyrazine in Rhodococcus jostii strain TMP1. By comparing protein expression profiles, we identified a tetramethylpyrazine-inducible protein of 40 kDa and determined its identity by tandem mass spectrometry (MS-MS) de novo sequencing. Searches against an R. jostii TMP1 genome database allowed the identification of the tetramethylpyrazine-inducible protein-coding gene. The tetramethylpyrazine-inducible gene was located within a 13-kb genome cluster, denominated the tetramethylpyrazine degradation (tpd) locus, that encoded eight proteins involved in tetramethylpyrazine catabolism. The genes from this cluster were cloned and transferred into tetramethylpyrazine-nondegrading Rhodococcus erythropolis strain SQ1. This allowed us to verify the function of the tpd locus, to isolate intermediate metabolites, and to reconstruct the catabolic pathway of tetramethylpyrazine. We report that the degradation of tetramethylpyrazine is a multistep process that includes initial oxidative aromatic-ring cleavage by tetramethylpyrazine oxygenase, TpdAB; subsequent hydrolysis by (Z)-N,N'-(but-2-ene-2,3-diyl)diacetamide hydrolase, TpdC; and further intermediate metabolite reduction by aminoalcohol dehydrogenase, TpdE. Thus, the genes responsible for bacterial degradation of pyrazines have been identified, and intermediate metabolites of tetramethylpyrazine degradation have been isolated for the first time.

  9. Plasmid localization and organization of melamine degradation genes in Rhodococcus sp. strain Mel.

    PubMed

    Dodge, Anthony G; Wackett, Lawrence P; Sadowsky, Michael J

    2012-03-01

    Rhodococcus sp. strain Mel was isolated from soil by enrichment and grew in minimal medium with melamine as the sole N source with a doubling time of 3.5 h. Stoichiometry studies showed that all six nitrogen atoms of melamine were assimilated. The genome was sequenced by Roche 454 pyrosequencing to 13× coverage, and a 22.3-kb DNA region was found to contain a homolog to the melamine deaminase gene trzA. Mutagenesis studies showed that the cyanuric acid hydrolase and biuret hydrolase genes were clustered together on a different 17.9-kb contig. Curing and gene transfer studies indicated that 4 of 6 genes required for the complete degradation of melamine were located on an ∼265-kb self-transmissible linear plasmid (pMel2), but this plasmid was not required for ammeline deamination. The Rhodococcus sp. strain Mel melamine metabolic pathway genes were located in at least three noncontiguous regions of the genome, and the plasmid-borne genes encoding enzymes for melamine metabolism were likely recently acquired.

  10. Combination of degradation pathways for naphthalene utilization in Rhodococcus sp. strain TFB

    PubMed Central

    Tomás-Gallardo, Laura; Gómez-Álvarez, Helena; Santero, Eduardo; Floriano, Belén

    2014-01-01

    Rhodococcus sp. strain TFB is a metabolic versatile bacterium able to grow on naphthalene as the only carbon and energy source. Applying proteomic, genetic and biochemical approaches, we propose in this paper that, at least, three coordinated but independently regulated set of genes are combined to degrade naphthalene in TFB. First, proteins involved in tetralin degradation are also induced by naphthalene and may carry out its conversion to salicylaldehyde. This is the only part of the naphthalene degradation pathway showing glucose catabolite repression. Second, a salicylaldehyde dehydrogenase activity that converts salicylaldehyde to salicylate is detected in naphthalene-grown cells but not in tetralin-or salicylate-grown cells. Finally, we describe the chromosomally located nag genes, encoding the gentisate pathway for salicylate conversion into fumarate and pyruvate, which are only induced by salicylate and not by naphthalene. This work shows how biodegradation pathways in Rhodococcus sp. strain TFB could be assembled using elements from different pathways mainly because of the laxity of the regulatory systems and the broad specificity of the catabolic enzymes. PMID:24325207

  11. Role of amine oxidase expression to maintain putrescine homeostasis in Rhodococcus opacus.

    PubMed

    Foster, Alexander; Barnes, Nicole; Speight, Robert; Morris, Peter C; Keane, Mark A

    2013-04-10

    While applications of amine oxidases are increasing, few have been characterised and our understanding of their biological role and strategies for bacteria exploitation are limited. By altering the nitrogen source (NH4Cl, putrescine and cadaverine (diamines) and butylamine (monoamine)) and concentration, we have identified a constitutive flavin dependent oxidase (EC 1.4.3.10) within Rhodococcus opacus. The activity of this oxidase can be increased by over two orders of magnitude in the presence of aliphatic diamines. In addition, the expression of a copper dependent diamine oxidase (EC 1.4.3.22) was observed at diamine concentrations>1mM or when cells were grown with butylamine, which acts to inhibit the flavin oxidase. A Michaelis-Menten kinetic treatment of the flavin oxidase delivered a Michaelis constant (KM)=190μM and maximum rate (kcat)=21.8s(-1) for the oxidative deamination of putrescine with a lower KM (=60μM) and comparable kcat (=18.2s(-1)) for the copper oxidase. MALDI-TOF and genomic analyses have indicated a metabolic clustering of functionally related genes. From a consideration of amine oxidase specificity and sequence homology, we propose a putrescine degradation pathway within Rhodococcus that utilises oxidases in tandem with subsequent dehydrogenase and transaminase enzymes. The implications of PUT homeostasis through the action of the two oxidases are discussed with respect to stressors, evolution and application in microbe-assisted phytoremediation or bio-augmentation.

  12. Plasmid Localization and Organization of Melamine Degradation Genes in Rhodococcus sp. Strain Mel

    PubMed Central

    Dodge, Anthony G.; Wackett, Lawrence P.

    2012-01-01

    Rhodococcus sp. strain Mel was isolated from soil by enrichment and grew in minimal medium with melamine as the sole N source with a doubling time of 3.5 h. Stoichiometry studies showed that all six nitrogen atoms of melamine were assimilated. The genome was sequenced by Roche 454 pyrosequencing to 13× coverage, and a 22.3-kb DNA region was found to contain a homolog to the melamine deaminase gene trzA. Mutagenesis studies showed that the cyanuric acid hydrolase and biuret hydrolase genes were clustered together on a different 17.9-kb contig. Curing and gene transfer studies indicated that 4 of 6 genes required for the complete degradation of melamine were located on an ∼265-kb self-transmissible linear plasmid (pMel2), but this plasmid was not required for ammeline deamination. The Rhodococcus sp. strain Mel melamine metabolic pathway genes were located in at least three noncontiguous regions of the genome, and the plasmid-borne genes encoding enzymes for melamine metabolism were likely recently acquired. PMID:22210223

  13. Combination of degradation pathways for naphthalene utilization in Rhodococcus sp. strain TFB.

    PubMed

    Tomás-Gallardo, Laura; Gómez-Álvarez, Helena; Santero, Eduardo; Floriano, Belén

    2014-03-01

    Rhodococcus sp. strain TFB is a metabolic versatile bacterium able to grow on naphthalene as the only carbon and energy source. Applying proteomic, genetic and biochemical approaches, we propose in this paper that, at least, three coordinated but independently regulated set of genes are combined to degrade naphthalene in TFB. First, proteins involved in tetralin degradation are also induced by naphthalene and may carry out its conversion to salicylaldehyde. This is the only part of the naphthalene degradation pathway showing glucose catabolite repression. Second, a salicylaldehyde dehydrogenase activity that converts salicylaldehyde to salicylate is detected in naphthalene-grown cells but not in tetralin- or salicylate-grown cells. Finally, we describe the chromosomally located nag genes, encoding the gentisate pathway for salicylate conversion into fumarate and pyruvate, which are only induced by salicylate and not by naphthalene. This work shows how biodegradation pathways in Rhodococcus sp. strain TFB could be assembled using elements from different pathways mainly because of the laxity of the regulatory systems and the broad specificity of the catabolic enzymes. © 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  14. [Respiratory infections caused by slow-growing bacteria: Nocardia, Actinomyces, Rhodococcus].

    PubMed

    Eschapasse, E; Hussenet, C; Bergeron, A; Lebeaux, D

    2017-06-01

    Pneumonia caused by slow-growing bacteria is rare but sometimes severe. These infections share many similarities such as several differential diagnoses, difficulties to identify the pathogen, the importance of involving the microbiologist in the diagnostic investigation and the need for prolonged antibiotic treatment. However, major differences distinguish them: Nocardia and Rhodococcus infect mainly immunocompromised patients while actinomycosis also concerns immunocompetent patients; the severity of nocardioses is related to their hematogenous spread while locoregional extension by contiguity makes the gravity of actinomycosis. For these diseases, molecular diagnostic tools are essential, either to obtain a species identification and guide treatment in the case of nocardiosis or to confirm the diagnosis from a biological sample. Treatment of these infections is complex due to: (1) the limited data in the literature; (2) the need for prolonged treatment of several months; (3) the management of toxicities and drug interactions for the treatment of Nocardia and Rhodococcus. Close cooperation between pneumonologists, infectious disease specialists and microbiologists is essential for the management of these patients. Copyright © 2017 SPLF. Published by Elsevier Masson SAS. All rights reserved.

  15. Media optimization for biosurfactant production by Rhodococcus erythropolis MTCC 2794: artificial intelligence versus a statistical approach.

    PubMed

    Pal, Moumita P; Vaidya, Bhalchandra K; Desai, Kiran M; Joshi, Renuka M; Nene, Sanjay N; Kulkarni, Bhaskar D

    2009-05-01

    This paper entails a comprehensive study on production of a biosurfactant from Rhodococcus erythropolis MTCC 2794. Two optimization techniques--(1) artificial neural network (ANN) coupled with genetic algorithm (GA) and (2) response surface methodology (RSM)--were used for media optimization in order to enhance the biosurfactant yield by Rhodococcus erythropolis MTCC 2794. ANN and RSM models were developed, incorporating the quantity of four medium components (sucrose, yeast extract, meat peptone, and toluene) as independent input variables and biosurfactant yield [calculated in terms of percent emulsification index (% EI(24))] as output variable. ANN-GA and RSM were compared for their predictive and generalization ability using a separate data set of 16 experiments, for which the average quadratic errors were approximately 3 and approximately 6%, respectively. ANN-GA was found to be more accurate and consistent in predicting optimized conditions and maximum yield than RSM. For the ANN-GA model, the values of correlation coefficient and average quadratic error were approximately 0.99 and approximately 3%, respectively. It was also shown that ANN-based models could be used accurately for sensitivity analysis. ANN-GA-optimized media gave about a 3.5-fold enhancement in biosurfactant yield.

  16. Physiological and genetic differences amongst Rhodococcus species for using glycerol as a source for growth and triacylglycerol production.

    PubMed

    Herrero, O Marisa; Moncalián, Gabriel; Alvarez, Héctor M

    2016-02-01

    We analysed the ability of five different rhodococcal species to grow and produce triacylglycerols (TAGs) from glycerol, the main byproduct of biodiesel production. Rhodococcus fascians and Rhodococcus erythropolis grew fast on glycerol, whereas Rhodococcus opacus and Rhodococcus jostii exhibited a prolonged lag phase of several days before growing. Rhodococcus equi only exhibited poor growth on glycerol. R. erythropolis DSMZ 43060 and R. fascians F7 produced 3.9-4.3 g cell biomass l(-1) and 28.4-44.6% cellular dry weight (CDW) of TAGs after 6 days of incubation; whereas R. opacus PD630 and R. jostii RHA1 produced 2.5-3.8 g cell biomass l(-1) and 28.3-38.4% CDW of TAGs after 17 days of growth on glycerol. Genomic analyses revealed two different sets of genes for glycerol uptake and degradation (here named clusters 1 and 2) amongst rhodococci. Those species that possessed cluster 1 (glpFK1D1) (R. fascians and R. erythropolis) exhibited fast growth and lipid accumulation, whereas those that possessed cluster 2 (glpK2D2) (R. opacus, R. jostii and R. equi) exhibited delayed growth and lipid accumulation during cultivation on glycerol. Three glycerol-negative strains were complemented for their ability to grow and produce TAGs by heterologous expression of glpK2 from R. opacus PD630. In addition, we significantly reduced the extension of the lag phase and improved glycerol assimilation and oil production of R. opacus PD630 when expressing glpK1D1 from R. fascians. The results demonstrated that rhodococci are a flexible and amenable biological system for further biotechnological applications based on the reutilization of glycerol.

  17. Cold adaptive traits revealed by comparative genomic analysis of the eurypsychrophile Rhodococcus sp. JG3 isolated from high elevation McMurdo Dry Valley permafrost, Antarctica.

    PubMed

    Goordial, Jacqueline; Raymond-Bouchard, Isabelle; Zolotarov, Yevgen; de Bethencourt, Luis; Ronholm, Jennifer; Shapiro, Nicole; Woyke, Tanja; Stromvik, Martina; Greer, Charles W; Bakermans, Corien; Whyte, Lyle

    2016-02-01

    The permafrost soils of the high elevation McMurdo Dry Valleys are the most cold, desiccating and oligotrophic on Earth. Rhodococcus sp. JG3 is one of very few bacterial isolates from Antarctic Dry Valley permafrost, and displays subzero growth down to -5°C. To understand how Rhodococcus sp. JG3 is able to survive extreme permafrost conditions and be metabolically active at subzero temperatures, we sequenced its genome and compared it to the genomes of 14 mesophilic rhodococci. Rhodococcus sp. JG3 possessed a higher copy number of genes for general stress response, UV protection and protection from cold shock, osmotic stress and oxidative stress. We characterized genome wide molecular adaptations to cold, and identified genes that had amino acid compositions favourable for increased flexibility and functionality at low temperatures. Rhodococcus sp. JG3 possesses multiple complimentary strategies which may enable its survival in some of the harshest permafrost on Earth.

  18. Rhodococcus rhodochrous ATCC12674 Becomes Alkane-Tolerant upon GroEL2 Overexpression and Survives in the n-Octane Phase in Two Phase Culture

    PubMed Central

    Takihara, Hayato; Matsuura, Chiaki; Ogihara, Jun; Iwabuchi, Noriyuki; Sunairi, Michio

    2014-01-01

    We recently reported that the overexpression of GroEL2 played an important role in increasing the alkane tolerance of Rhodococcus erythropolis PR4. In the present study, we examined the effects of the introduction of groEL2 on the alkane tolerance of other Rhodococcus strains. The introduction of groEL2 into Rhodococcus strains led to increased alkane tolerance. The translocation of R. rhodochrous ATCC12674 cells to and survival in the n-octane (C8) phase in two phase culture were significantly enhanced by the introduction of groEL2 derived from strain PR4, suggesting that engineering cells to overexpress GroEL2 represents an effective strategy for enhancing organic solvent tolerance in Rhodococcus. PMID:25491752

  19. Whole-Genome Shotgun Sequencing of Rhodococcus erythropolis Strain P27, a Highly Radiation-Resistant Actinomycete from Antarctica

    PubMed Central

    Gouvêa Taketani, Rodrigo; Domingues Zucchi, Tiago; Soares de Melo, Itamar

    2013-01-01

    Here, we report the draft genome sequence of radiation-resistant Rhodococcus erythropolis strain P27, isolated from leaves of Deschampsia antarctica Desv. (Poaceae) in the Admiralty Bay area, Antarctica. PMID:24072865

  20. Cloning of the genes for degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine from Rhodococcus sp. strain TE1.

    PubMed Central

    Shao, Z Q; Behki, R

    1995-01-01

    The degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine (2-chloro-4-ethyl-amino-6-isopropylamino-1,3,5-triazine) is associated with an indigenous plasmid in Rhodococcus sp. strain TE1. Plasmid DNA libraries of Rhodococcus sp. strain TE1 were constructed in a Rhodococcus-Escherichia coli shuttle vector, pBS305, and transferred into Rhodococcus sp. strain TE3, a derivative of Rhodococcus sp. strain TE1 lacking herbicide degradation activity, to select transformants capable of growing on EPTC as the sole source of carbon (EPTC+). Analysis of plasmids from the EPTC+ transformants indicated that the eptA gene, which codes for the enzyme required for EPTC degradation, residues on a 6.2-kb KpnI fragment. The cloned fragment also harbored the gene required for atrazine N dealkylation (atrA). The plasmid carrying the cloned fragment could be electroporated into a number of other Rhodococcus strains in which both eptA and atrA were fully expressed. No expression of the cloned genes was evident in E. coli strains. Subcloning of the 6.2-kb fragment to distinguish between EPTC- and atrazine-degrading genes was not successful. PMID:7646050

  1. Simultaneous species-specific PCR detection and viability testing of poly(vinyl alcohol) cryogel-entrapped Rhodococcus spp. after their exposure to petroleum hydrocarbons.

    PubMed

    Kuyukina, Maria S; Ivshina, Irena B; Serebrennikova, Marina K; Rubtsova, Ekaterina V; Krivoruchko, Anastasiya V

    2013-08-01

    A method of simultaneous species-specific PCR detection and viability testing of poly(vinyl alcohol) cryogel-entrapped Rhodococcus spp. was developed that allowed the estimation of immobilized Rhodococcus opacus and Rhodococcus ruber survival after their exposure to petroleum hydrocarbon mixture. Spectrophotometric INT assay revealed high tolerance of gel-immobilized rhodococci to petroleum hydrocarbons, while among two Rhodococcus strains studied, R. ruber tolerated better to hydrocarbons compared to R. opacus. These findings were confirmed by respirometry results that showed increased respiratory activity of gel-immobilized Rhodococcus strains after 10-day incubation with 3% (v/v) petroleum hydrocarbon mixture. Moreover, jointly incubated rhodococcal strains demonstrated higher oxidative activities toward petroleum hydrocarbons than individual strains. Both Rhodococcus species were recovered successfully in cryogel granules using 16S rDNA-targeted PCR, even though the granules were previously stained with INT and extracted with ethanol. The method developed can be used for rapid detection and monitoring of gel-immobilized bacterial inocula in bioreactors or contaminated soil systems. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Effect of Aromatic Compounds on Cellular Fatty Acid Composition of Rhodococcus opacus

    PubMed Central

    Tsitko, Irina V.; Zaitsev, Gennadi M.; Lobanok, Anatoli G.; Salkinoja-Salonen, Mirja S.

    1999-01-01

    In cells of Rhodococcus opacus GM-14, GM-29, and 1CP, the contents of branched (10-methyl) fatty acids increased from 3% to 15 to 34% of the total fatty acids when the cells were grown on benzene, phenol, 4-chlorophenol, chlorobenzene, or toluene as the sole source of carbon and energy, in comparison with cells grown on fructose. In addition, the content of trans-hexadecenoic acid increased from 5% to 8 to 18% with phenol or chlorophenol as the carbon source. The 10-methyl branched fatty acid content of R. opacus GM-14 cells increased in a dose-related manner following exposure to phenol or toluene when toluene was not utilized as the growth substrate. The results suggest that 10-methyl branched fatty acids may participate in the adaptation of R. opacus to lipophilic aromatic compounds. PMID:9925629

  3. Formaldehyde removal in synthetic and industrial wastewater by Rhodococcus erythropolis UPV-1.

    PubMed

    Hidalgo, A; Lopategi, A; Prieto, M; Serra, J L; Llama, M J

    2002-02-01

    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.

  4. A real-time impedance based method to assess Rhodococcus equi virulence.

    PubMed

    Miranda-CasoLuengo, Aleksandra A; Miranda-CasoLuengo, Raúl; Lieggi, Nora T; Luo, Haixia; Simpson, Jeremy C; Meijer, Wim G

    2013-01-01

    Rhodococcus equi is a facultative intracellular pathogen of macrophages and the causative agent of foal pneumonia. R. equi virulence is usually assessed by analyzing intracellular growth in macrophages by enumeration of bacteria following cell lysis, which is time consuming and does not allow for a high throughput analysis. This paper describes the use of an impedance based real-time method to characterize proliferation of R. equi in macrophages, using virulent and attenuated strains lacking the vapA gene or virulence plasmid. Image analysis suggested that the time-dependent cell response profile (TCRP) is governed by cell size and roundness as well as cytoxicity of infecting R. equi strains. The amplitude and inflection point of the resulting TCRP were dependent on the multiplicity of infection as well as virulence of the infecting strain, thus distinguishing between virulent and attenuated strains.

  5. Equid herpesvirus 1 and rhodococcus equi coinfection in a foal with bronchointerstitial pneumonia.

    PubMed

    Perez-Ecija, Alejandro; Mendoza, Francisco Javier; Estepa, José Carlos; Bautista, María José; Pérez, José

    2016-10-01

    A 2-month-old foal with septic shock and severe respiratory distress was referred to the Veterinary Teaching Hospital. Due to poor prognosis, the foal was euthanized. Histopathology showed lesions suggestive of Rhodococcus equi infection associated with a diffuse interstitial infiltrate of foamy macrophages and syncytial cells presenting large acidophilic intranuclear inclusion bodies, fibrin exudates and hyaline membranes. Bacteriological examination from lung and respiratory exudates confirmed R. equi infection, whilst immunohistochemistry and PCR yielded a positive result for Equid herpesvirus type 1 (EHV-1). Several etiologies have been proposed for bronchointerstitial pneumonia in foals, although a multifactorial origin for this lesional pattern could be possible. This work is the first one describing a combined EHV-1 and R. equi infection in a foal affected with bronchointerstitial pneumonia.

  6. A Real-Time Impedance Based Method to Assess Rhodococcus equi Virulence

    PubMed Central

    Lieggi, Nora T.; Luo, Haixia; Simpson, Jeremy C.; Meijer, Wim G.

    2013-01-01

    Rhodococcus equi is a facultative intracellular pathogen of macrophages and the causative agent of foal pneumonia. R. equi virulence is usually assessed by analyzing intracellular growth in macrophages by enumeration of bacteria following cell lysis, which is time consuming and does not allow for a high throughput analysis. This paper describes the use of an impedance based real-time method to characterize proliferation of R. equi in macrophages, using virulent and attenuated strains lacking the vapA gene or virulence plasmid. Image analysis suggested that the time-dependent cell response profile (TCRP) is governed by cell size and roundness as well as cytoxicity of infecting R. equi strains. The amplitude and inflection point of the resulting TCRP were dependent on the multiplicity of infection as well as virulence of the infecting strain, thus distinguishing between virulent and attenuated strains. PMID:23555995

  7. Immune Reconstitution Syndrome secondary to Rhodococcus equi infection in a patient with HIV and Burkitt's lymphoma.

    PubMed

    Darraj, Majid; Fainstein, Rachel; Kasper, Ken; Keynan, Yoav

    Immune Reconstitution Syndrome (IRIS) has been associated with a variety of infections in patients with human immunodeficiency virus (HIV). However, we are reporting the first case of IRIS secondary to Rhodococcus equi (R. equi) in a patient with HIV. We report the case of a 48-year-old male found to have HIV infection in the setting of Burkitt's lymphoma. While on anti-retroviral therapy and chemotherapy, he had developed IRIS secondary to R. equi that manifested as a cavitating pneumonia. This report outlines the successful management of the R. equi infection with the use of a combination of antibiotics, radiographic follow up and suppressive antibiotic while on chemotherapy. Copyright © 2016 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

  8. Rhodococcus equi pneumonia in the foal--part 1: pathogenesis and epidemiology.

    PubMed

    Muscatello, Gary

    2012-04-01

    Rhodococcus equi pneumonia is a worldwide infectious disease of major concern to the equine breeding industry. The disease typically manifests in foals as pyogranulomatous bronchopneumonia, resulting in significant morbidity and mortality. Inhalation of aerosolised virulent R. equi from the environment and intracellular replication within alveolar macrophages are essential components of the pathogenesis of R. equi pneumonia in the foal. Recently documented evidence of airborne transmission between foals indicates the potential for an alternative contagious route of disease transmission. In the first of this two-part review, the complexity of the host, pathogen and environmental interactions that underpin R. equi pneumonia will be discussed through an exploration of current understanding of the epidemiology and pathogenesis of R. equi pneumonia in the foal. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. Rhodococcus equi human clinical isolates enter and survive within human alveolar epithelial cells.

    PubMed

    Ramos-Vivas, J; Pilares-Ortega, L; Remuzgo-Martínez, S; Padilla, D; Gutiérrez-Díaz, J L; Navas-Méndez, J

    2011-05-01

    Rhodococcus equi is an emerging opportunistic human pathogen associated with immunosuppressed people, especially those infected with the human immunodeficiency virus (HIV). This pathogen resides primarily within lung macrophages of infected patients, which may explain in part its ability to escape normal pulmonary defense mechanisms. Despite numerous studies as a pulmonary pathogen in foals, where a plasmid seems to play an important role in virulence, information on the pathogenesis of this pathogen in humans is still scarce. In this study, fluorescence microscopy and vancomycin protection assays were used to investigate the ability of R. equi human isolates to adhere to and to invade the human alveolar epithelial cell line A549. Our findings indicate that some R. equi clinical strains are capable of adhering, entering and surviving within the alveolar cell line, which may contribute to the pathogen persistence in lung tissues. Copyright © 2011 Institut Pasteur. Published by Elsevier SAS. All rights reserved.

  10. Antibiotic failure in a renal transplant patient with Rhodococcus equi infection: an indication for surgical lobectomy.

    PubMed

    Ursales, A; Klein, J A; Beal, S G; Koch, M; Clement-Kruzel, S; Melton, L B; Spak, C W

    2014-12-01

    Rhodococcus equi is an animal pathogen that causes infrequent but challenging infections in immunocompromised individuals, few of which have been described in solid organ transplant recipients. Common clinical presentations include indolent cough, fever, and dyspnea, with necrotizing pneumonia and cavitation. We report a case of a dense right upper lung pneumonia with resultant R. equi bacteremia in a renal transplant recipient. Our patient initially responded to antibiotic treatment with resolution of bacteremia and clinical recovery, followed by interval progression in her right upper lobe consolidation on follow-up computed tomography scans. She underwent lobectomy for definitive therapy with resolution of symptoms. Lobectomy can be utilized in isolated infection after antibiotic failure with excellent clinical outcomes. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. Tuberculosis-like pneumonias by the aerobic actinomycetes Rhodococcus, Tsukamurella and Gordonia.

    PubMed

    Savini, Vincenzo; Fazii, Paolo; Favaro, Marco; Astolfi, Daniela; Polilli, Ennio; Pompilio, Arianna; Vannucci, Mariangela; D'Amario, Claudio; Di Bonaventura, Giovanni; Fontana, Carla; D'Antonio, Domenico

    2012-05-01

    The order Actinomycetales includes phylogenetically diverse but morphologically similar aerobic and anaerobic organisms, exhibiting filamentous branching structures which fragment into rods or coccoid forms. Lung pathogens of the order comprise Mycobacterium, Nocardia, Corynebacterium, Actinomyces, Kytococcus, Rothia, Williamsia, as well as Gordonia, Tsukamurella and Rhodococcus. Particularly, members of the last three genera are uncommon aerobic agents of lung cavitations and tuberculosis(TB)-like syndromes, that should be carefully considered in the aetiology of parenchymal lesions. Correct identification of such organisms is hard to obtain, but is crucial to provide patients with adequate diagnose and treatment. Then, this review aims to unearth their airway tropism, as well as their clinical impact as agents of lung disease. Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  12. The plant pathogen Rhodococcus fascians colonizes the exterior and interior of the aerial parts of plants.

    PubMed

    Cornelis, K; Ritsema, T; Nijsse, J; Holsters, M; Goethals, K; Jaziri, M

    2001-05-01

    Rhodococcus fascians is a plant-pathogenic bacterium that causes malformations on aerial plant parts, whereby leafy galls occur at axillary meristems. The colonization behavior on Nicotiana tabacum and Arabidopsis thaliana plants was examined. Independent of the infection methods, R. fascians extensively colonized the plant surface where the bacteria were surrounded by a slime layer. R. fascians caused the collapse of epidermal cells and penetrated intercellularly into the plant tissues. The onset of symptom development preceded the extensive colonization of the interior. The meristematic regions induced by pathogenic strain D188 were surrounded by bacteria. The nonpathogenic strain, D188-5, colonized the exterior of the plant equally well, but the linear plasmid (pFiD188) seemed to be involved in the penetration efficiency and colonization of tobacco tissues.

  13. A Review on The Bioconversion of Lignin to Microbial Lipid with Oleaginous Rhodococcus opacus

    DOE PAGES

    Mahan, Kristina M.; Le, Rosemary K.; Yuan, Joshua; ...

    2017-06-29

    Rhodococcus opacus produces intracellular lipids from the biodegradation of lignocellulosic biomass. These lipids can be used to produce biofuels that could potentially replace petroleum-derived chemicals. Some current studies are focusing on deconstructing lignin through efficient and cost-effective pretreatment methods and improving microbial lipid titers. Furthermore, R. opacus can reach high levels of oleaginicity (>80%) when grown on glucose and other aromatic model compounds but intracellular lipid production is much lower on complex recalcitrant lignin substrates. Our review will discuss recent advances in studying R. opacus lignin degradation by exploring different pretreatment methods, increasing lignin solubility, enriching for low molecular weightmore » lignin compounds and laccase supplementation.« less

  14. Purification and properties of an amidase from Rhodococcus erythropolis MP50 which enantioselectively hydrolyzes 2-arylpropionamides.

    PubMed Central

    Hirrlinger, B; Stolz, A; Knackmuss, H J

    1996-01-01

    An enantioselective amidase from Rhodococcus erythropolis MP50 was purified to homogeneity. The enzyme has a molecular weight of about 480,000 and is composed of identical subunits with molecular weights of about 61,000. The NH2-terminal amino acid sequence was significantly different from previously published sequences of bacterial amidases. The purified amidase hydrolyzed a wide range of aliphatic and aromatic amides, The highest enzyme activities were found with amides carrying hydrophobic residues, such as pentyl or naphthoyl. The purified enzyme converted racemic 2-phenylpropionamide, naproxen amide [2-(6-methoxy-2-naphthyl) propionamide], and ketoprofen amide [2-(3'-benzoylphenyl)propionamide] to the corresponding S-acids with an enantiomeric excess of >99% and an almost 50% conversion of the racemic amides. The enzyme also hydrolyzed different alpha-amino amides but without significant enantioselectivity. PMID:8655547

  15. The fas operon of Rhodococcus fascians encodes new genes required for efficient fasciation of host plants.

    PubMed

    Crespi, M; Vereecke, D; Temmerman, W; Van Montagu, M; Desomer, J

    1994-05-01

    Three virulence loci (fas, att, and hyp) of Rhodococcus fascians D188 have been identified on a 200-kb conjugative linear plasmid (pFiD188). The fas locus was delimited to a 6.5-kb DNA fragment by insertion mutagenesis, single homologous disruptive recombination, and in trans complementation of different avirulent insertion mutants. The locus is arranged as a large operon containing six open reading frames whose expression is specifically induced during the interaction with host plants. One predicted protein is homologous to P-450 cytochromes from actinomycetes. The putative ferredoxin component is of a novel type containing additional domains homologous to transketolases from chemoautotrophic, photosynthetic, and methylotrophic microorganisms. Genetic analysis revealed that fas encodes, in addition to the previously identified ipt, at least two new genes that are involved in fasciation development, one of which is only required on older tobacco plants.

  16. A successful bacterial coup d'état: how Rhodococcus fascians redirects plant development.

    PubMed

    Stes, Elisabeth; Vandeputte, Olivier M; El Jaziri, Mondher; Holsters, Marcelle; Vereecke, Danny

    2011-01-01

    Rhodococcus fascians is a gram-positive phytopathogen that induces differentiated galls, known as leafy galls, on a wide variety of plants, employing virulence genes located on a linear plasmid. The pathogenic strategy consists of the production of a mixture of six synergistically acting cytokinins that overwhelm the plant's homeostatic mechanisms, ensuring the activation of a signaling cascade that targets the plant cell cycle and directs the newly formed cells to differentiate into shoot meristems. The shoots that are formed upon infection remain immature and never convert to source tissues resulting in the establishment of a nutrient sink that is a niche for the epiphytic and endophytic R. fascians subpopulations. Niche formation is accompanied by modifications of the transcriptome, metabolome, physiology, and morphology of both host and pathogen. Here, we review a decade of research and set the outlines of the molecular basis of the leafy gall syndrome.

  17. Molecular biology of coal bio-desulfurization. [Rhodococcus rhodochrous, Desulfovibrio desulfuricans

    SciTech Connect

    Young, K.D.; Gallagher, J.R.

    1992-04-30

    Genes cloned from Rhodococcus rhodochrous IGTS8 can transfer the DBT desulfurization phenotype to a different species (R. Fascians). The product was identified as 2-phenylphenol by gas chromatography. This result parallels the results we have previously reported for the activity of these genes in a DBT-negative mutant of IGTS8. Thus, the evidence is strong that we have identified and cloned the entire set of genes that are responsible for this very specific desulfurization reaction. Sequencing of these genes has commenced. A genomic library was constructed from the bacterium, Besulfovibrio desulfuricans. Screening has not yet identified a clone that carries the desulfurization genes from that organism. Two open reading frames, doxH and doxJ, in the C18 DBT degradation pathway were mutated and are now believed to be dispensable to that pathway. Finally, progress was made toward beginning to sequence the DBT dixoygenase genes from strain A15.

  18. Removal of sulfur-containing organic molecules adsorbed on inorganic supports by Rhodococcus Rhodochrous spp.

    PubMed

    Carvajal, P; Dinamarca, M Alejandro; Baeza, P; Camú, E; Ojeda, J

    2017-02-01

    To remove dibenzothiophene (DBT) and 4,6-dimethyl-dibenzothiophene (4,6-DMDBT) adsorbed on alumina, silica and sepiolite through biodesulfurization (BDS) using Rhodococcus Rhodochrous spp., that selectively reduce sulfur molecules without generating of gaseous pollutants. The adsorption of DBT and 4,6-DMDBT was affected by the properties of the supports, including particle size and the presence of surface acidic groups. The highest adsorption of both sulfur-containing organic molecules used particle sizes of 0.43-0.063 mm. The highest percentage removal was with sepiolite (80 % for DBT and 56 % for 4,6-DMDBT) and silica (71 % for DBT and 37 % for 4,6-DMDBT). This is attributed to the close interaction between these supports and the bacteria. Biodesulfurization is effective for removing the sulfur-containing organic molecules adsorbed on inorganic materials and avoids the generation of gaseous pollutants.

  19. Bioconversion of acrylonitrile to acrylamide using polyacrylamide entrapped cells of Rhodococcus rhodochrous PA-34.

    PubMed

    Raj, J; Prasad, S; Sharma, N N; Bhalla, T C

    2010-09-01

    The nitrile hydratase (NHase) of Rhodococcus rhodochrous PA-34 catalyzed the conversion of acrylonitrile to acrylamide. The resting cells (having NHase activity) (8 %; 1 mL corresponds to 22 mg dry cell mass, DCM) were immobilized in polyacrylamide gel containing 12.5 % acrylamide, 0.6 % bisacrylamide, 0.2 % diammonium persulfate and 0.4 % TEMED. The polyacrylamide entrapped cells (1.12 mg DCM/mL) completely converted acrylonitrile in 3 h at 10 °C, using 0.1 mol/L potassium phosphate buffer. In a partitioned fed batch reactor, 432 g/L acrylamide was accumulated after 1 d. The polyacrylamide discs were recycled up to 3×; 405, 210 and 170 g/L acrylamide was produced in 1st, 2nd and 3rd recycling reactions. In four cycles, a total of 1217 g acrylamide was produced by recycling the same mass of entrapped cells.

  20. In vitro antimicrobial activity of gallium maltolate against virulent Rhodococcus equi.

    PubMed

    Coleman, Michelle; Kuskie, Kyle; Liu, Mei; Chaffin, Keith; Libal, Melissa; Giguère, Steeve; Bernstein, Lawrence; Cohen, Noah

    2010-11-20

    The objective of this study was to determine the in vitro antimicrobial activity of gallium maltolate (GaM) against Rhodococcus equi. A total of 98 virulent bacterial isolates from equine clinical cases were examined, of which 19 isolates were known to be resistant to macrolides and rifampin. Isolates were cultured with various concentrations of GaM and minimal inhibitory concentration (MIC) values were determined after 24 and 48 h. Both the MIC(50) and the MIC(90) after 24h of growth were 558 ng/mL (8 μM) and after 48 h of growth were 2230 ng/mL (32 μM). There were no apparent differences between MICs of macrolide-resistant and macrolide-susceptible isolates.

  1. Desulfurization and denitrogenation of heavy gas oil by Rhodococcus erythropolis ATCC 4277.

    PubMed

    Maass, D; Todescato, D; Moritz, D E; Oliveira, J Vladimir; Oliveira, D; Ulson de Souza, A A; Guelli Souza, S M A

    2015-08-01

    Some of the noxious atmospheric pollutants such as nitrogen and sulfur dioxides come from the fossil fuel combustion. Biodesulfurization and biodenitrogenation are processes which remove those pollutants through the action of microorganisms. The ability of sulfur and nitrogen removal by the strain Rhodococcus erythropolis ATCC 4277 was tested in a biphasic system containing different heavy gas oil concentrations in a batch reactor. Heavy gas oil is an important fraction of petroleum, because after passing through, the vacuum distillation is incorporated into diesel oil. This strain was able to remove about 40% of the nitrogen and sulfur present in the gas heavy oil. Additionally, no growth inhibition occurred even when in the presence of pure heavy gas oil. Results present in this work are considered relevant for the development of biocatalytic processes for nitrogen and sulfur removal toward building feasible industrial applications.

  2. Equid herpesvirus 1 and rhodococcus equi coinfection in a foal with bronchointerstitial pneumonia

    PubMed Central

    PEREZ-ECIJA, Alejandro; MENDOZA, Francisco Javier; ESTEPA, José Carlos; BAUTISTA, María José; PÉREZ, José

    2016-01-01

    A 2-month-old foal with septic shock and severe respiratory distress was referred to the Veterinary Teaching Hospital. Due to poor prognosis, the foal was euthanized. Histopathology showed lesions suggestive of Rhodococcus equi infection associated with a diffuse interstitial infiltrate of foamy macrophages and syncytial cells presenting large acidophilic intranuclear inclusion bodies, fibrin exudates and hyaline membranes. Bacteriological examination from lung and respiratory exudates confirmed R. equi infection, whilst immunohistochemistry and PCR yielded a positive result for Equid herpesvirus type 1 (EHV-1). Several etiologies have been proposed for bronchointerstitial pneumonia in foals, although a multifactorial origin for this lesional pattern could be possible. This work is the first one describing a combined EHV-1 and R. equi infection in a foal affected with bronchointerstitial pneumonia. PMID:27264610

  3. Substrate Preferences in Biodesulfurization of Diesel Range Fuels by Rhodococcus sp. Strain ECRD-1

    PubMed Central

    Prince, Roger C.; Grossman, Matthew J.

    2003-01-01

    The range of sulfur compounds in fuel oil and the substrate range and preference of the biocatalytic system determine the maximum extent to which sulfur can be removed by biodesulfurization. We show that the biodesulfurization apparatus in Rhodococcus sp. strain ECRD-1 is able to attack all isomers of dibenzothiophene including those with at least four pendant carbons, with a slight preference for those substituted in the α-position. With somewhat less avidity, this apparatus is also able to attack substituted benzothiophenes with between two and seven pendant carbons. Some compounds containing sulfidic sulfur are also susceptible to desulfurization, although we have not yet been able to determine their molecular identities. PMID:14532032

  4. Protection of foals against experimental Rhodococcus equi pneumonia by oral immunization.

    PubMed Central

    Chirino-Trejo, J M; Prescott, J F; Yager, J A

    1987-01-01

    Two groups of three one to three week old foals were immunized orally on four occasions over five weeks with two strains of Rhodococcus equi, a clinical isolate from a pneumonic foal and a laboratory passaged Congo red negative variant of this strain. Three nonimmunized foals of similar age acted as controls. Three weeks after the last immunization, all foals were challenged on five occasions over seven days by aerosol infection with about 10(10) of the pneumonic foal isolate on each occasion. Control foals became seriously ill and were euthanized. Immunization with either strain protected foals equally against the challenge, and resulted in rapid lung clearance. Oral immunization can thus protect foals against severe challenge with R. equi. The proteins associated with Congo red colony staining appear not to be involved in protective immunity. PMID:3453264

  5. Recovery of Rhodococcus biosurfactants using methyl tertiary-butyl ether extraction.

    PubMed

    Kuyukina, M S; Ivshina, I B; Philp, J C; Christofi, N; Dunbar, S A; Ritchkova, M I

    2001-08-01

    In the present study, we proposed methyl tertiary-butyl ether (MTBE) as a solvent for extraction of biosurfactants from Rhodococcus bacterial cultures. After comparison with other well known solvent systems used for biosurfactant extraction, it was found that MTBE was able to extract crude surfactant material with high product recovery (10 g/l), efficiency (critical micelle concentration (CMC), 130-170 mg/l) and good functional surfactant characteristics (surface and interfacial tensions, 29 and 0.9 mN/m, respectively). The isolated surfactant complex contained 10% polar lipids, mostly glycolipids possessing maximal surface activity. Ultrasonic treatment of the extraction mixture increased the proportion of polar lipids in crude extract, resulting in increasing surfactant efficiency. Due to certain characteristics of MTBE, such as relatively low toxicity, biodegradability, ease of downstream recovery, low flammability and explosion safety, the use of this solvent as an extraction agent in industrial scale biosurfactant production is feasible.

  6. Nitrile Hydratase and Amidase from Rhodococcus rhodochrous Hydrolyze Acrylic Fibers and Granular Polyacrylonitriles

    PubMed Central

    Tauber, M. M.; Cavaco-Paulo, A.; Robra, K.-H.; Gübitz, G. M.

    2000-01-01

    Rhodococcus rhodochrous NCIMB 11216 produced nitrile hydratase (320 nkat mg of protein−1) and amidase activity (38.4 nkat mg of protein−1) when grown on a medium containing propionitrile. These enzymes were able to hydrolyze nitrile groups of both granular polyacrylonitriles (PAN) and acrylic fibers. Nitrile groups of PAN40 (molecular mass, 40 kDa) and PAN190 (molecular mass, 190 kDa) were converted into the corresponding carbonic acids to 1.8 and 1.0%, respectively. In contrast, surfacial nitrile groups of acrylic fibers were only converted to the corresponding amides. X-ray photoelectron spectroscopy analysis showed that 16% of the surfacial nitrile groups were hydrolyzed by the R. rhodochrous enzymes. Due to the enzymatic modification, the acrylic fibers became more hydrophilic and thus, adsorption of dyes was enhanced. This was indicated by a 15% increase in the staining level (K/S value) for C.I. Basic Blue 9. PMID:10742253

  7. Biodegradation of the Organic Disulfide 4,4′-Dithiodibutyric Acid by Rhodococcus spp.

    PubMed Central

    Khairy, Heba; Wübbeler, Jan Hendrik

    2015-01-01

    Four Rhodococcus spp. exhibited the ability to use 4,4′-dithiodibutyric acid (DTDB) as a sole carbon source for growth. The most important step for the production of a novel polythioester (PTE) using DTDB as a precursor substrate is the initial cleavage of DTDB. Thus, identification of the enzyme responsible for this step was mandatory. Because Rhodococcus erythropolis strain MI2 serves as a model organism for elucidation of the biodegradation of DTDB, it was used to identify the genes encoding the enzymes involved in DTDB utilization. To identify these genes, transposon mutagenesis of R. erythropolis MI2 was carried out using transposon pTNR-TA. Among 3,261 mutants screened, 8 showed no growth with DTDB as the sole carbon source. In five mutants, the insertion locus was mapped either within a gene coding for a polysaccharide deacetyltransferase, a putative ATPase, or an acetyl coenzyme A transferase, 1 bp upstream of a gene coding for a putative methylase, or 176 bp downstream of a gene coding for a putative kinase. In another mutant, the insertion was localized between genes encoding a putative transcriptional regulator of the TetR family (noxR) and an NADH:flavin oxidoreductase (nox). Moreover, in two other mutants, the insertion loci were mapped within a gene encoding a hypothetical protein in the vicinity of noxR and nox. The interruption mutant generated, R. erythropolis MI2 noxΩtsr, was unable to grow with DTDB as the sole carbon source. Subsequently, nox was overexpressed and purified, and its activity with DTDB was measured. The specific enzyme activity of Nox amounted to 1.2 ± 0.15 U/mg. Therefore, we propose that Nox is responsible for the initial cleavage of DTDB into 2 molecules of 4-mercaptobutyric acid (4MB). PMID:26407888

  8. The Hydroxamate Siderophore Rhequichelin Is Required for Virulence of the Pathogenic Actinomycete Rhodococcus equi

    PubMed Central

    Coulson, Garry B.; Miranda-CasoLuengo, Aleksandra; Vázquez-Boland, José A.; Hondalus, Mary K.

    2012-01-01

    We previously showed that the facultative intracellular pathogen Rhodococcus equi produces a nondiffusible and catecholate-containing siderophore (rhequibactin) involved in iron acquisition during saprophytic growth. Here, we provide evidence that the rhbABCDE cluster directs the biosynthesis of a hydroxamate siderophore, rhequichelin, that plays a key role in virulence. The rhbC gene encodes a nonribosomal peptide synthetase that is predicted to produce a tetrapeptide consisting of N5-formyl-N5-hydroxyornithine, serine, N5-hydroxyornithine, and N5-acyl-N5-hydroxyornithine. The other rhb genes encode putative tailoring enzymes mediating modification of ornithine residues incorporated into the hydroxamate product of RhbC. Transcription of rhbC was upregulated during growth in iron-depleted medium, suggesting that it plays a role in iron acquisition. This was confirmed by deletion of rhbCD, rendering the resulting strain R. equi SID2 unable to grow in the presence of the iron chelator 2,2-dipyridyl. Supernatant of the wild-type strain rescued the phenotype of R. equi SID2. The importance of rhequichelin in virulence was highlighted by the rapid increase in transcription levels of rhbC following infection and the inability of R. equi SID2 to grow within macrophages. Unlike the wild-type strain, R. equi SID2 was unable to replicate in vivo and was rapidly cleared from the lungs of infected mice. Rhequichelin is thus a key virulence-associated factor, although nonpathogenic Rhodococcus species also appear to produce rhequichelin or a structurally closely related compound. Rhequichelin biosynthesis may therefore be considered an example of cooption of a core actinobacterial trait in the evolution of R. equi virulence. PMID:22966042

  9. The Genome of a Pathogenic Rhodococcus: Cooptive Virulence Underpinned by Key Gene Acquisitions

    PubMed Central

    Letek, Michal; González, Patricia; MacArthur, Iain; Rodríguez, Héctor; Freeman, Tom C.; Valero-Rello, Ana; Blanco, Mónica; Buckley, Tom; Cherevach, Inna; Fahey, Ruth; Hapeshi, Alexia; Holdstock, Jolyon; Leadon, Desmond; Navas, Jesús; Ocampo, Alain; Quail, Michael A.; Sanders, Mandy; Scortti, Mariela M.; Prescott, John F.; Fogarty, Ursula; Meijer, Wim G.; Parkhill, Julian; Bentley, Stephen D.; Vázquez-Boland, José A.

    2010-01-01

    We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid–rich intestine and manure of herbivores—two main R. equi reservoirs. Except for a few horizontally acquired (HGT) pathogenicity loci, including a cytoadhesive pilus determinant (rpl) and the virulence plasmid vap pathogenicity island (PAI) required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche–adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT–acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi. PMID:20941392

  10. The hydroxamate siderophore rhequichelin is required for virulence of the pathogenic actinomycete Rhodococcus equi.

    PubMed

    Miranda-Casoluengo, Raúl; Coulson, Garry B; Miranda-Casoluengo, Aleksandra; Vázquez-Boland, José A; Hondalus, Mary K; Meijer, Wim G

    2012-12-01

    We previously showed that the facultative intracellular pathogen Rhodococcus equi produces a nondiffusible and catecholate-containing siderophore (rhequibactin) involved in iron acquisition during saprophytic growth. Here, we provide evidence that the rhbABCDE cluster directs the biosynthesis of a hydroxamate siderophore, rhequichelin, that plays a key role in virulence. The rhbC gene encodes a nonribosomal peptide synthetase that is predicted to produce a tetrapeptide consisting of N(5)-formyl-N(5)-hydroxyornithine, serine, N(5)-hydroxyornithine, and N(5)-acyl-N(5)-hydroxyornithine. The other rhb genes encode putative tailoring enzymes mediating modification of ornithine residues incorporated into the hydroxamate product of RhbC. Transcription of rhbC was upregulated during growth in iron-depleted medium, suggesting that it plays a role in iron acquisition. This was confirmed by deletion of rhbCD, rendering the resulting strain R. equi SID2 unable to grow in the presence of the iron chelator 2,2-dipyridyl. Supernatant of the wild-type strain rescued the phenotype of R. equi SID2. The importance of rhequichelin in virulence was highlighted by the rapid increase in transcription levels of rhbC following infection and the inability of R. equi SID2 to grow within macrophages. Unlike the wild-type strain, R. equi SID2 was unable to replicate in vivo and was rapidly cleared from the lungs of infected mice. Rhequichelin is thus a key virulence-associated factor, although nonpathogenic Rhodococcus species also appear to produce rhequichelin or a structurally closely related compound. Rhequichelin biosynthesis may therefore be considered an example of cooption of a core actinobacterial trait in the evolution of R. equi virulence.

  11. Enzymatic cyanide degradation by cell-free extract of Rhodococcus UKMP-5M.

    PubMed

    Nallapan Maniyam, Maegala; Sjahrir, Fridelina; Latif Ibrahim, Abdul; Cass, Anthony E G

    2015-01-01

    The cell-free extract of locally isolated Rhodococcus UKMP-5M strain was used as an alternative to develop greener and cost effective cyanide removal technology. The present study aims to assess the viability of the cell-free extract to detoxify high concentrations of cyanide which is measured through the monitoring of protein concentration and specific cyanide-degrading activity. When cyanide-grown cells were subjected to grinding in liquid nitrogen which is relatively an inexpressive and fast cell disruption method, highest cyanide-degrading activity of 0.63 mM min(-1) mg(-1) protein was obtained in comparison to enzymatic lysis and agitation with fine glass beads. The cell-free extracts managed to degrade 80% of 20 mM KCN within 80 min and the rate of cyanide consumption increased linearly as the concentration of protein was raised. In both cases, the addition of co-factor was not required which proved to be advantageous economically. The successful formation of ammonia and formate as endproducts indicated that the degradation of cyanide by Rhodococcus UKMP-5M proceeded via the activity of cyanidase and the resulting non-toxic products are safe for disposal into the environment. Further verification with SDS-PAGE revealed that the molecular weight of the active enzyme was estimated to be 38 kDa, which is consistent with previously reported cyanidases. Thus, the utilization of cell-free extracts as an alternative to live microbial in cyanide degradation offers numerous advantageous such as the potential to tolerate and degrade higher concentration of cyanide and total reduction in the overall cost of operation since the requirement for nutrient support is irrelevant.

  12. Utilization of simultaneous saccharification and fermentation residues as feedstock for lipid accumulation in Rhodococcus opacus.

    PubMed

    Le, Rosemary K; Das, Parthapratim; Mahan, Kristina M; Anderson, Seth A; Wells, Tyrone; Yuan, Joshua S; Ragauskas, Arthur J

    2017-09-29

    Use of oleaginous microorganisms as "micro-factories" for accumulation of single cell oils for biofuel production has increased significantly to mitigate growing energy demands, resulting in efforts to upgrade industrial waste, such as second-generation lignocellulosic residues, into potential feedstocks. Dilute-acid pretreatment (DAP) is commonly used to alter the physicochemical properties of lignocellulosic materials and is typically coupled with simultaneous saccharification and fermentation (SSF) for conversion of sugars into ethanol. The resulting DAP residues are usually processed as a waste stream, e.g. burned for power, but this provides minimal value. Alternatively, these wastes can be utilized as feedstock to generate lipids, which can be converted to biofuel. DAP-SSF residues were generated from pine, poplar, and switchgrass. High performance liquid chromatography revealed less than 0.13% monomeric sugars in the dry residue. Fourier transform infrared spectroscopy was indicative of the presence of lignin and polysaccharides. Gel permeation chromatography suggested the bacterial strains preferred molecules with molecular weight ~ 400-500 g/mol. DAP-SSF residues were used as the sole carbon source for lipid production by Rhodococcus opacus DSM 1069 and PD630 in batch fermentations. Depending on the strain of Rhodococcus employed, 9-11 lipids for PD630 and DSM 1069 were observed, at a final concentration of ~ 15 mg/L fatty acid methyl esters (FAME) detected. Though the DAP-SSF substrate resulted in low FAME titers, novel analysis of solid-state fermentations was investigated, which determined that DAP-SSF residues could be a viable feedstock for lipid generation.

  13. Genome-based exploration of the specialized metabolic capacities of the genus Rhodococcus.

    PubMed

    Ceniceros, Ana; Dijkhuizen, Lubbert; Petrusma, Mirjan; Medema, Marnix H

    2017-08-09

    Bacteria of the genus Rhodococcus are well known for their ability to degrade a large range of organic compounds. Some rhodococci are free-living, saprophytic bacteria; others are animal and plant pathogens. Recently, several studies have shown that their genomes encode putative pathways for the synthesis of a large number of specialized metabolites that are likely to be involved in microbe-microbe and host-microbe interactions. To systematically explore the specialized metabolic potential of this genus, we here performed a comprehensive analysis of the biosynthetic coding capacity across publicly available rhododoccal genomes, and compared these with those of several Mycobacterium strains as well as that of their mutual close relative Amycolicicoccus subflavus. Comparative genomic analysis shows that most predicted biosynthetic gene cluster families in these strains are clade-specific and lack any homology with gene clusters encoding the production of known natural products. Interestingly, many of these clusters appear to encode the biosynthesis of lipopeptides, which may play key roles in the diverse environments were rhodococci thrive, by acting as biosurfactants, pathogenicity factors or antimicrobials. We also identified several gene cluster families that are universally shared among all three genera, which therefore may have a more 'primary' role in their physiology. Inactivation of these clusters by mutagenesis might help to generate weaker strains that can be used as live vaccines. The genus Rhodococcus thus provides an interesting target for natural product discovery, in view of its large and mostly uncharacterized biosynthetic repertoire, its relatively fast growth and the availability of effective genetic tools for its genomic modification.

  14. The genome of a pathogenic rhodococcus: cooptive virulence underpinned by key gene acquisitions.

    PubMed

    Letek, Michal; González, Patricia; Macarthur, Iain; Rodríguez, Héctor; Freeman, Tom C; Valero-Rello, Ana; Blanco, Mónica; Buckley, Tom; Cherevach, Inna; Fahey, Ruth; Hapeshi, Alexia; Holdstock, Jolyon; Leadon, Desmond; Navas, Jesús; Ocampo, Alain; Quail, Michael A; Sanders, Mandy; Scortti, Mariela M; Prescott, John F; Fogarty, Ursula; Meijer, Wim G; Parkhill, Julian; Bentley, Stephen D; Vázquez-Boland, José A

    2010-09-30

    We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid-rich intestine and manure of herbivores--two main R. equi reservoirs. Except for a few horizontally acquired (HGT) pathogenicity loci, including a cytoadhesive pilus determinant (rpl) and the virulence plasmid vap pathogenicity island (PAI) required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche-adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT-acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi.

  15. Structure of the virulence-associated protein VapD from the intracellular pathogen Rhodococcus equi

    SciTech Connect

    Whittingham, Jean L.; Blagova, Elena V.; Finn, Ciaran E.; Luo, Haixia; Miranda-CasoLuengo, Raúl; Turkenburg, Johan P.; Leech, Andrew P.; Walton, Paul H.; Murzin, Alexey G.; Meijer, Wim G.; Wilkinson, Anthony J.

    2014-08-01

    VapD is one of a set of highly homologous virulence-associated proteins from the multi-host pathogen Rhodococcus equi. The crystal structure reveals an eight-stranded β-barrel with a novel fold and a glycine rich ‘bald’ surface. Rhodococcus equi is a multi-host pathogen that infects a range of animals as well as immune-compromised humans. Equine and porcine isolates harbour a virulence plasmid encoding a homologous family of virulence-associated proteins associated with the capacity of R. equi to divert the normal processes of endosomal maturation, enabling bacterial survival and proliferation in alveolar macrophages. To provide a basis for probing the function of the Vap proteins in virulence, the crystal structure of VapD was determined. VapD is a monomer as determined by multi-angle laser light scattering. The structure reveals an elliptical, compact eight-stranded β-barrel with a novel strand topology and pseudo-twofold symmetry, suggesting evolution from an ancestral dimer. Surface-associated octyl-β-d-glucoside molecules may provide clues to function. Circular-dichroism spectroscopic analysis suggests that the β-barrel structure is preceded by a natively disordered region at the N-terminus. Sequence comparisons indicate that the core folds of the other plasmid-encoded virulence-associated proteins from R. equi strains are similar to that of VapD. It is further shown that sequences encoding putative R. equi Vap-like proteins occur in diverse bacterial species. Finally, the functional implications of the structure are discussed in the light of the unique structural features of VapD and its partial structural similarity to other β-barrel proteins.

  16. Web-Type Evolution of Rhodococcus Gene Clusters Associated with Utilization of Naphthalene

    PubMed Central

    Kulakov, Leonid A.; Chen, Shenchang; Allen, Christopher C. R.; Larkin, Michael J.

    2005-01-01

    Clusters of genes which include determinants for the catalytic subunits of naphthalene dioxygenase (narAa and narAb) were analyzed in naphthalene-degrading Rhodococcus strains. We demonstrated (i) that in the region analyzed homologous gene clusters are separated from each other by nonhomologous DNA, (ii) that there are various degrees of homology between related genes, and (iii) that nar genes are located on plasmids in strains NCIMB12038 and P400 and on a chromosome in P200. These observations suggest that genetic exchange and reshuffling of genetic modules, as well as vertical descent of the genetic information, were the main routes in the evolution of naphthalene degradation in Rhodococcus. These conclusions were supported by studies of transcription patterns in the region analyzed. It was found that the nar region is not organized into a single operon but there are several transcription units which differ in the strains investigated. The narA and narB genes were found to be transcribed as a single unit in all strains analyzed, and their transcription was induced by naphthalene. The putative aldolase gene (narC) was found on the same transcript only in strains P200 and P400. In NCIMB12038 transcription of two more gene clusters was induced by growth on naphthalene. Transcription start sites for narA and narB were found to be different in all of the strains studied. Putative regulatory genes (narR1 and narR2) were transcribed as a single mRNA in naphthalene-induced cells. At the same time, a number of the genes known to be essential for naphthalene catabolism in gram-negative bacteria were not found in the region analyzed. PMID:15811998

  17. [Effects of nitriles and amides on the growth and the nitrile hydratase activity of the Rhodococcus sp. strain gt1].

    PubMed

    Maksimov, A Iu; Kuznetsova, M V; Ovechkina, G V; Kozlov, S V; Maksimova, Iu G; Demakov, V A

    2003-01-01

    Effects of some nitriles and amides, as well as glucose and ammonium, on the growth and the nitrile hydratase (EC 4.2.1.84) activity of the Rhodococcus sp. strain gt1 isolated from soil were studied. The activity of nitrile hydratase mainly depended on carbon and nitrogen supply to cells. The activity of nitrile hydratase was high in the presence of glucose and ammonium at medium concentrations and decreased at concentrations of glucose more than 0.3%. Saturated unsubstituted aliphatic nitriles and amides were found to be a good source of nitrogen and carbon. However, the presence of nitriles and amides in the medium was not absolutely necessary for the expression of the activity of nitrile hydratase isolated from the Rhodococcus sp. strain gt1.

  18. Overexpression of a phosphatidic acid phosphatase type 2 leads to an increase in triacylglycerol production in oleaginous Rhodococcus strains.

    PubMed

    Hernández, Martín A; Comba, Santiago; Arabolaza, Ana; Gramajo, Hugo; Alvarez, Héctor M

    2015-03-01

    Oleaginous Rhodococcus strains are able to accumulate large amounts of triacylglycerol (TAG). Phosphatidic acid phosphatase (PAP) enzyme catalyzes the dephosphorylation of phosphatidic acid (PA) to yield diacylglycerol (DAG), a key precursor for TAG biosynthesis. Studies to establish its role in lipid metabolism have been mainly focused in eukaryotes but not in bacteria. In this work, we identified and characterized a putative PAP type 2 (PAP2) encoded by the ro00075 gene in Rhodococcus jostii RHA1. Heterologous expression of ro00075 in Escherichia coli resulted in a fourfold increase in PAP activity and twofold in DAG content. The conditional deletion of ro00075 in RHA1 led to a decrease in the content of DAG and TAG, whereas its overexpression in both RHA1 and Rhodococcus opacus PD630 promoted an increase up to 10 to 15 % by cellular dry weight in TAG content. On the other hand, expression of ro00075 in the non-oleaginous strain Rhodococcus fascians F7 promoted an increase in total fatty acid content up to 7 % at the expense of free fatty acid (FFA), DAG, and TAG fractions. Moreover, co-expression of ro00075/atf2 genes resulted in a fourfold increase in total fatty acid content by a further increase of the FFA and TAG fractions. The results of this study suggest that ro00075 encodes for a PAP2 enzyme actively involved in TAG biosynthesis. Overexpression of this gene, as single one or with an atf gene, provides an alternative approach to increase the biosynthesis and accumulation of bacterial oils as a potential source of raw material for biofuel production.

  19. Isolation and identification of berberine and berberrubine metabolites by berberine-utilizing bacterium Rhodococcus sp. strain BD7100.

    PubMed

    Ishikawa, Kazuki; Takeda, Hisashi; Wakana, Daigo; Sato, Fumihiko; Hosoe, Tomoo

    2016-05-01

    Based on the finding of a novel berberine (BBR)-utilizing bacterium, Rhodococcus sp. strain BD7100, we investigated the degradation of BBR and its analog berberrubine (BRU). Resting cells of BD7100 demethylenated BBR and BRU, yielding benzeneacetic acid analogs. Isolation of benzeneacetic acid analogs suggested that BD7100 degraded the isoquinoline ring of the protoberberine skeleton. This work represents the first report of cleavage of protoberberine skeleton by a microorganism.

  20. Isolation and characterization of a Rhodococcus strain with phenol-degrading ability and its potential use for tannery effluent biotreatment.

    PubMed

    Paisio, Cintia E; Talano, Melina A; González, Paola S; Busto, Víctor D; Talou, Julián Rodríguez; Agostini, Elizabeth

    2012-09-01

    Wastewater derived from leather production may contain phenols, which are highly toxic, and their degradation could be possible through bioremediation technologies. In the present work, microbial degradation of phenol was studied using a tolerant bacterial strain, named CS1, isolated from tannery sediments. This strain was able to survive in the presence of phenol at concentrations of up to 1,000 mg/L. On the basis of morphological and biochemical properties, 16S rRNA gene sequencing, and phylogenetic analysis, the isolated strain was identified as Rhodococcus sp. Phenol removal was evaluated at a lab-scale in Erlenmeyer flasks and at a bioreactor scale in a stirred tank reactor. Rhodococcus sp. CS1 was able to completely remove phenol in a range of 200 to 1,000 mg/L in mineral medium at 30 ± 2 °C and pH 7 as optimal conditions. In the stirred tank bioreactor, we studied the effect of some parameters, such as agitation (200-600 rpm) and aeration (1-3 vvm), on growth and phenol removal efficiency. Faster phenol biodegradation was obtained in the bioreactor than in Erlenmeyer flasks, and maximum phenol removal was achieved at 400 rpm and 1 vvm in only 12 h. Furthermore, Rhodococcus sp. CS1 strain was able to grow and completely degrade phenols from tannery effluents after 9 h of incubation. Based on these results, Rhodococcus sp. CS1 could be an appropriate microorganism for bioremediation of tannery effluents or other phenol-containing wastewaters.

  1. Characterization of carbon-sulfur bond cleavage by axenic and mixed cultures of Rhodococcus rhodochrous IGTS8

    SciTech Connect

    Kayser, K.J.; Bielaga, B.A.; Jackowski, K.; Oduson, O.; Kilbane, J. II

    1992-12-31

    Growth assays reveal that Rhodococcus rhodochrous IGTS8 can utilize a wide range of organosulfur compounds as the sole source of sulfur. Compounds that are utilized include thiophenes, sulfides, disulfides, mercaptans, sulfoxides, and sulfones. None of the organosulfur compounds tested can serve as a carbon source. A convenient spectrophotometric assay (Gibbs assay) based on the chromogenic reaction of 2,6-dichloroquinone-4-chloroimide with aromatic hydroxyl groups was developed and used in conjunction with GC/MS analysis to examine the kinetics of carbon-sulfur bond cleavage by axenic and mixed cell cultures of Rhodococcus rhodochrous IGTS8. The desulfurization trait is expressed at uniform levels during the mid-exponential phase, reaches a maximum during idiophase, and then declines in stationary-phase cells. Desulfurization rates for dibenzothiophene (DBT) range from 8 to 15 {mu}M of DBT/10{sup 12} cells/hour. Mixtures of genetically marked Rhodococcus rhodochrous IGTS8 and an organisms incapable of cleaning carbon-sulfur bonds in relevant test compounds, Enterobacter cloacae, were prepared in ratios that varied over six orders of magnitude. Growth studies revealed that Enterobacter cloacae was able to gain access to sulfur liberated from organosulfur compounds by IGTS8; however, cell-to-cell contact was required. These data also indicate that the desulfurization activity of IGTS8 cells in mixed cultures may be as much as 200-fold higher than in axenic cultures.

  2. Microaerophilic degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by three Rhodococcus strains.

    PubMed

    Fuller, M E; Perreault, N; Hawari, J

    2010-09-01

    The goal of this study was to compare the degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by three Rhodococcus strains under anaerobic, microaerophilic (<0.04 mg l(-1) dissolved oxygen) and aerobic (dissolved oxygen (DO) maintained at 8 mg l(-1)) conditions. Three Rhodococcus strains were incubated with no, low and ambient concentrations of oxygen in minimal media with succinate as the carbon source and RDX as the sole nitrogen source. RDX and RDX metabolite concentrations were measured over time. Under microaerophilic conditions, the bacteria degraded RDX, albeit about 60-fold slower than under fully aerobic conditions. Only the breakdown product, 4-nitro-2,4-diazabutanal (NDAB) accumulated to measurable concentrations under microaerophilic conditions. RDX degraded quickly under both aerated and static aerobic conditions (DO allowed to drop below 1 mg l(-1)) with the accumulation of both NDAB and methylenedinitramine (MEDINA). No RDX degradation was observed under strict anaerobic conditions. The Rhodococcus strains did not degrade RDX under strict anaerobic conditions, while slow degradation was observed under microaerophilic conditions. The RDX metabolite NDAB was detected under both microaerophilic and aerobic conditions, while MEDINA was detected only under aerobic conditions. IMPACT AND SIGNIFICANCE OF THE STUDY: This work confirmed the production of MEDINA under aerobic conditions, which has not been previously associated with aerobic RDX degradation by these organisms. More importantly, it demonstrated that aerobic rhodococci are able to degrade RDX under a broader range of oxygen concentrations than previously reported.

  3. Triacylglycerol accumulation and oxidative stress in Rhodococcus species: differential effects of pro-oxidants on lipid metabolism.

    PubMed

    Urbano, Susana Bequer; Di Capua, Cecilia; Cortez, Néstor; Farías, María E; Alvarez, Héctor M

    2014-03-01

    In general, members of Rhodococcus genus are highly resistant to desiccation. Desiccation is a complex process which includes the formation of reactive oxygen species that results in significant damage to cells. In this study, we demonstrate that extremophile actinobacterial strains isolated from diverse environments, mainly belonging to Rhodococcus genus, exhibited high tolerance to the pro-oxidants hydrogen peroxide (H2O2) and methyl viologen (MV). In addition, we investigated the possible interconnections between the responses of the oleaginous Rhodococcus opacus PD630 to oxidative stress and lipid metabolism, since both processes demand a metabolic reorganization of cells. Experiments with metabolic inhibitors showed differential effects of both pro-oxidants on lipid metabolism in PD630 cells. The inhibition of carotenoid biosynthesis by the addition of diphenylamine to the media negatively affected the tolerance of cells to H2O2, but not to MV. The inhibition of triacylglycerol (TAG) biosynthesis and accumulation in PD630 did not affect the tolerance of cells to H2O2 and MV; whereas, the blockage of lipolysis decreased the tolerance of cells to H2O2 (but not MV) under carbon-starvation conditions. Interestingly, the addition of MV to the media (but not H2O2) induced a reduction of TAG accumulation by cells. Resuming, results of this study revealed metabolic connections between lipid metabolism and oxidative stress responses in R. opacus PD630, and probably in other extremophile TAG-accumulating rhodococci.

  4. Analysis and optimization of triacylglycerol synthesis in novel oleaginous Rhodococcus and Streptomyces strains isolated from desert soil.

    PubMed

    Röttig, Annika; Hauschild, Philippa; Madkour, Mohamed H; Al-Ansari, Ahmed M; Almakishah, Naief H; Steinbüchel, Alexander

    2016-05-10

    As oleaginous microorganisms represent an upcoming novel feedstock for the biotechnological production of lipids or lipid-derived biofuels, we searched for novel, lipid-producing strains in desert soil. This was encouraged by the hypothesis that neutral lipids represent an ideal storage compound, especially under arid conditions, as several animals are known to outlast long periods in absence of drinking water by metabolizing their body fat. Ten lipid-accumulating bacterial strains, affiliated to the genera Bacillus, Cupriavidus, Nocardia, Rhodococcus and Streptomyces, were isolated from arid desert soil due to their ability to synthesize poly(β-hydroxybutyrate), triacylglycerols or wax esters. Particularly two Streptomyces sp. strains and one Rhodococcus sp. strain accumulate significant amounts of TAG under storage conditions under optimized cultivation conditions. Rhodococcus sp. A27 and Streptomyces sp. G49 synthesized approx. 30% (w/w) fatty acids from fructose or cellobiose, respectively, while Streptomyces isolate G25 reached a cellular fatty acid content of nearly 50% (w/w) when cultivated with cellobiose. The stored triacylglycerols were composed of 30-40% branched fatty acids, such as anteiso-pentadecanoic or iso-hexadecanoic acid. To date, this represents by far the highest lipid content described for streptomycetes. A biotechnological production of such lipids using (hemi)cellulose-derived raw material could be used to obtain sustainable biodiesel with a high proportion of branched-chain fatty acids to improve its cold-flow properties and oxidative stability.

  5. A Rhodococcus qsdA-Encoded Enzyme Defines a Novel Class of Large-Spectrum Quorum-Quenching Lactonases▿ †

    PubMed Central

    Uroz, Stéphane; Oger, Phil M.; Chapelle, Emilie; Adeline, Marie-Thérèse; Faure, Denis; Dessaux, Yves

    2008-01-01

    A gene involved in N-acyl homoserine lactone (N-AHSL) degradation was identified by screening a genomic library of Rhodococcus erythropolis strain W2. This gene, named qsdA (for quorum-sensing signal degradation), encodes an N-AHSL lactonase unrelated to the two previously characterized N-AHSL-degrading enzymes, i.e., the lactonase AiiA and the amidohydrolase AiiD. QsdA is related to phosphotriesterases and constitutes the reference of a novel class of N-AHSL degradation enzymes. It confers the ability to inactivate N-AHSLs with an acyl chain ranging from C6 to C14, with or without substitution at carbon 3. Screening of a collection of 15 Rhodococcus strains and strains closely related to this genus clearly highlighted the relationship between the ability to degrade N-AHSLs and the presence of the qsdA gene in Rhodococcus. Bacteria harboring the qsdA gene interfere very efficiently with quorum-sensing-regulated functions, demonstrating that qsdA is a valuable tool for developing quorum-quenching procedures. PMID:18192419

  6. Influence of Plasmid Type on the Replication of Rhodococcus equi in Host Macrophages

    PubMed Central

    Willingham-Lane, Jennifer M.; Berghaus, Londa J.; Giguère, Steeve

    2016-01-01

    ABSTRACT The soil-dwelling, saprophytic actinomycete Rhodococcus equi is a multihost, facultative intracellular pathogen of macrophages. When inhaled by susceptible foals, it causes severe bronchopneumonia. It is also a pathogen of pigs, which may develop submaxillary lymphadenitis upon exposure. R. equi isolates obtained from foals and pigs possess conjugative plasmids housing a pathogenicity island (PAI) containing a novel family of genes of unknown function called the virulence-associated protein or vap family. The PAI regions of the equine and swine plasmids differ in vap gene composition, with equine isolates possessing six vap genes, including the major virulence determinant vapA, while the PAIs of swine isolates house vapB and five other unique vap genes. Possession of the pVAPA-type virulence plasmid by equine isolates bestows the capacity for intramacrophage replication essential for disease development in vivo. Swine isolates of R. equi are largely unstudied. Here, we show that R. equi isolates from pigs, carrying pVAPB-type plasmids, are able to replicate in a plasmid-dependent manner in macrophages obtained from a variety of species (murine, swine, and equine) and anatomical locations. Similarly, equine isolates carrying pVAPA-type plasmids are capable of replication in swine macrophages. Plasmid swapping between equine and swine strains through conjugation did not alter the intracellular replication capacity of the parental strain, indicating that coevolution of the plasmid and chromosome is not crucial for this attribute. These results demonstrate that while distinct plasmid types exist among R. equi isolates obtained from equine and swine sources, this tropism is not determined by host species-specific intramacrophage replication capabilities. IMPORTANCE This work greatly advances our understanding of the opportunistic pathogen Rhodococcus equi, a disease agent of animals and immunocompromised people. Clinical isolates from diseased foals carry a

  7. Influence of Plasmid Type on the Replication of Rhodococcus equi in Host Macrophages.

    PubMed

    Willingham-Lane, Jennifer M; Berghaus, Londa J; Giguère, Steeve; Hondalus, Mary K

    2016-01-01

    The soil-dwelling, saprophytic actinomycete Rhodococcus equi is a multihost, facultative intracellular pathogen of macrophages. When inhaled by susceptible foals, it causes severe bronchopneumonia. It is also a pathogen of pigs, which may develop submaxillary lymphadenitis upon exposure. R. equi isolates obtained from foals and pigs possess conjugative plasmids housing a pathogenicity island (PAI) containing a novel family of genes of unknown function called the virulence-associated protein or vap family. The PAI regions of the equine and swine plasmids differ in vap gene composition, with equine isolates possessing six vap genes, including the major virulence determinant vapA, while the PAIs of swine isolates house vapB and five other unique vap genes. Possession of the pVAPA-type virulence plasmid by equine isolates bestows the capacity for intramacrophage replication essential for disease development in vivo. Swine isolates of R. equi are largely unstudied. Here, we show that R. equi isolates from pigs, carrying pVAPB-type plasmids, are able to replicate in a plasmid-dependent manner in macrophages obtained from a variety of species (murine, swine, and equine) and anatomical locations. Similarly, equine isolates carrying pVAPA-type plasmids are capable of replication in swine macrophages. Plasmid swapping between equine and swine strains through conjugation did not alter the intracellular replication capacity of the parental strain, indicating that coevolution of the plasmid and chromosome is not crucial for this attribute. These results demonstrate that while distinct plasmid types exist among R. equi isolates obtained from equine and swine sources, this tropism is not determined by host species-specific intramacrophage replication capabilities. IMPORTANCE This work greatly advances our understanding of the opportunistic pathogen Rhodococcus equi, a disease agent of animals and immunocompromised people. Clinical isolates from diseased foals carry a

  8. Biosurfactants of Rhodococcus erythropolis IMV Ас-5017: synthesis intensification and practical application.

    PubMed

    Pirog, Tetyana; Sofilkanych, Anna; Shevchuk, Tetyana; Shulyakova, Mariya

    2013-06-01

    Intensification of the surfactant synthesis by Rhodococcus erythropolis IMV Ac-5017 on different substrates, including industrial waste, as well as the use of surfactant preparations for oil degradation were studied. It was established that the addition of fumarate (0.2 %) and citrate (0.1 %) into the medium with ethanol, n-hexadecane, or glycerol (1-2 %) was accompanied by an increase of conditional surfactant concentration by 1.5-1.7 times compared to the indexes in the medium without organic acids. The intensification of surfactant synthesis in the presence of fumarate and citrate is caused by the increased activity of isocitrate lyase (by 1.2-15-fold) and enzymes of the surfactant biosynthesis (by 2-4.8-fold) compared to their activity in the medium without precursors. The possibility of surfactant synthesis intensification (by 3-4-fold) while cultivating of R. erythropolis IMV Ac-5017 in the medium with oil containing substrates (2 %) and glucose (0.1 %) was shown. The introduction of 0.01 mM Cu(2+) in the exponential growth phase of strain IMV Ac-5017 in the medium with ethanol accompanied by the increasing conditional surfactant concentration by 1.9 times. The highly efficient remediation (92-95 %) of oil (2-2.6 g/L) and Cu(2+) polluted water after treatment with surfactant preparations (native cultural liquid) at low concentrations (5 %) was determined.

  9. Degradation of Chloronitrobenzenes by a Coculture of Pseudomonas putida and a Rhodococcus sp.

    PubMed Central

    Park, Hee-Sung; Lim, Sung-Jin; Chang, Young Keun; Livingston, Andrew G.; Kim, Hak-Sung

    1999-01-01

    A single microorganism able to mineralize chloronitrobenzenes (CNBs) has not been reported, and degradation of CNBs by coculture of two microbial strains was attempted. Pseudomonas putida HS12 was first isolated by analogue enrichment culture using nitrobenzene (NB) as the substrate, and this strain was observed to possess a partial reductive pathway for the degradation of NB. From high-performance liquid chromatography-mass spectrometry and 1H nuclear magnetic resonance analyses, NB-grown cells of P. putida HS12 were found to convert 3- and 4-CNBs to the corresponding 5- and 4-chloro-2-hydroxyacetanilides, respectively, by partial reduction and subsequent acetylation. For the degradation of CNBs, Rhodococcus sp. strain HS51, which degrades 4- and 5-chloro-2-hydroxyacetanilides, was isolated and combined with P. putida HS12 to give a coculture. This coculture was confirmed to mineralize 3- and 4-CNBs in the presence of an additional carbon source. A degradation pathway for 3- and 4-CNBs by the two isolated strains was also proposed. PMID:10049867

  10. A new acylamidase from Rhodococcus erythropolis TA37 can hydrolyze N-substituted amides.

    PubMed

    Lavrov, K V; Zalunin, I A; Kotlova, E K; Yanenko, A S

    2010-08-01

    A new acylamidase was isolated from Rhodococcus erythropolis TA37 and characterized. N-Substituted acrylamides (isopropyl acrylamide, N,N-dimethyl-aminopropyl acrylamide, and methylene-bis-acrylamide), acid para-nitroanilides (4'-nitroacetanilide, Gly-pNA, Ala-pNA, Leu-pNA), and N-acetyl derivatives of glycine, alanine, and leucine are good substrates for this enzyme. Aliphatic amides (acetamide, acrylamide, isobutyramide, n-butyramide, and valeramide) are also used as substrates but with less efficiency. The enzyme subunit mass by SDS-PAGE is 55 kDa. Maximal activity is exhibited at pH 7-8 and 55°C. The enzyme is stable for 15 h at 22°C and for 0.5 h at 45°C. The Michaelis constant (K(m)) is 0.25 mM with Gly-pNA and 0.55 mM with Ala-pNA. The acylamidase activity is suppressed by inhibitors of serine proteases (phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate) but is not suppressed by inhibitors of aliphatic amidases (acetaldehyde and nitrophenyl disulfides). The N-terminal amino acid sequence of the acylamidase is highly homologous to those of two putative amidases detected from sequenced R. erythropolis genomes. It is suggested that the acylamidase together with the detected homologs forms a new class within the amidase signature family.

  11. Calcium carbonate formation on mica supported extracellular polymeric substance produced by Rhodococcus opacus

    NASA Astrophysics Data System (ADS)

    Szcześ, Aleksandra; Czemierska, Magdalena; Jarosz-Wilkołazka, Anna

    2016-10-01

    Extracellular polymeric substance (EPS) extracted from Rhodococcus opacus bacterial strain was used as a matrix for calcium carbonate precipitation using the vapour diffusion method. The total exopolymer and water-soluble exopolymer fraction of different concentrations were spread on the mica surface by the spin-coating method. The obtained layers were characterized using the atomic force microscopy measurement and XPS analysis. The effects of polymer concentration, initial pH of calcium chloride solution and precipitation time on the obtained crystals properties were investigated. Raman spectroscopy and scanning electron microscopy were used to characterize the precipitated minerals. It was found that the type of precipitated CaCO3 polymorph and the crystal size depend on the kind of EPS fraction. The obtained results indicates that the water soluble fraction favours vaterite dissolution and calcite growth, whereas the total EPS stabilizes vaterite and this effect is stronger at basic pH. It seems to be due to different contents of the functional group of EPS fractions.

  12. Disseminated rhodococcus equi infection in HIV infection despite highly active antiretroviral therapy

    PubMed Central

    2011-01-01

    Background Rhodococcus equi (R.equi) is an acid fast, GRAM + coccobacillus, which is widespread in the soil and causes pulmonary and extrapulmonary infections in immunocompromised people. In the context of HIV infection, R.equi infection (rhodococcosis) is regarded as an opportunistic disease, and its outcome is influenced by highly active antiretroviral therapy (HAART). Case presentation We report two cases of HIV-related rhodococcosis that disseminated despite suppressive HAART and anti-rhodococcal treatment; in both cases there was no immunological recovery, with CD4+ cells count below 200/μL. In the first case, pulmonary rhodococcosis presented 6 months after initiation of HAART, and was followed by an extracerebral intracranial and a cerebral rhodococcal abscess 1 and 8 months, respectively, after onset of pulmonary infection. The second case was characterized by a protracted course with spread of infection to various organs, including subcutaneous tissue, skin, colon and other intra-abdominal tissues, and central nervous system; the spread started 4 years after clinical resolution of a first pulmonary manifestation and progressed over a period of 2 years. Conclusions Our report highlights the importance of an effective immune recovery, despite fully suppressive HAART, along with anti-rhodococcal therapy, in order to clear rhodococcal infection. PMID:22168333

  13. Identification, characterization and molecular analysis of the viable but nonculturable Rhodococcus biphenylivorans

    PubMed Central

    Su, Xiaomei; Sun, Faqian; Wang, Yalin; Hashmi, Muhammad Zaffar; Guo, Li; Ding, Linxian; Shen, Chaofeng

    2015-01-01

    Numerous bacteria, including pollutant-degrading bacteria can enter the viable but nonculturable state (VBNC) when they encounter harsh environmental conditions. VBNC bacteria, as a vast majority of potent microbial resource can be of great significance in environmental rehabilitation. It is necessary to study the VBNC state of pollutant-degrading bacteria under various stress conditions. The aim of this study was to determine whether Rhodococcus biphenylivorans could enter the VBNC state under oligotrophic and low temperature conditions, and to examine the changes of morphology, enzymatic activity and gene expressions that might underline such state. The obtained results indicated that R. biphenylivorans TG9T could enter into the VBNC state and recover culturability under favorable environmental conditions. Results from Illumina high throughput RNA-sequencing revealed that the up-regulated genes related to ATP accumulation, protein modification, peptidoglycan biosynthesis and RNA polymerase were found in the VBNC cells, and the down-regulated genes mainly encoded hypothetical protein, membrane protein and NADH dehydrogenase subunit, which render VBNC cells more tolerant to survive under inhospitable conditions. This study provides new insights into prevention and control of the VBNC state of pollutant-degrading bacteria for their better capabilities in environmental rehabilitation. PMID:26687808

  14. The Rhodococcus equi virulence protein VapA disrupts endolysosome function and stimulates lysosome biogenesis.

    PubMed

    Rofe, Adam P; Davis, Luther J; Whittingham, Jean L; Latimer-Bowman, Elizabeth C; Wilkinson, Anthony J; Pryor, Paul R

    2017-04-01

    Rhodococcus equi (R. equi) is an important pulmonary pathogen in foals that often leads to the death of the horse. The bacterium harbors a virulence plasmid that encodes numerous virulence-associated proteins (Vaps) including VapA that is essential for intracellular survival inside macrophages. However, little is known about the precise function of VapA. Here, we demonstrate that VapA causes perturbation to late endocytic organelles with swollen endolysosome organelles having reduced Cathepsin B activity and an accumulation of LBPA, LC3 and Rab7. The data are indicative of a loss of endolysosomal function, which leads cells to upregulate lysosome biogenesis to compensate for the loss of functional endolysosomes. Although there is a high degree of homology of the core region of VapA to other Vap proteins, only the highly conserved core region of VapA, and not VapD of VapG, gives the observed effects on endolysosomes. This is the first demonstration of how VapA works and implies that VapA aids R. equi survival by reducing the impact of lysosomes on phagocytosed bacteria. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  15. [Rhodococcus equi infection in AIDS patients: retrospective analysis of 13 patients in Argentina].

    PubMed

    Corti, Marcelo; Solari, Rubén; De Carolis, Luis; Palmieri, Omar; Rollet, Raquel; Shah, Haroun N

    2014-08-01

    Rhodococcus equi is a gram positive coccoid rod that causes pulmonary infections in immunosuppressed patients. We retrospectively analyzed epidemiological, clinical, microbiological, radiological, and immunological features as well as the outcomes of 13 AIDS patients with R. equi infection. Between January 1994 and December 2012, 13 patients attending the AIDS department of the Infectious Diseases reference hospital in Buenos Aires were diagnosed with R. equi infection. All were men, the median age was 27 years. At the time of diagnosis, the median of CD4+ T cell counts was 11 cells/μl Twelve patients presented pulmonary disease with isolation of the microorganism from sputum or bronchoalveolar lavage; in the other patient the diagnosis was postmortem with positive culture of cerebrospinal fluid. The most frequent clinical manifestations were fever, haemoptysis, and weight loss. The predominant radiological finding was lobe consolidation with cavitation. Nine patients died after a median survival of 5.5 months. In all of them, cultures persisted positive until the last admission. The other 4 patients did continue clinical follow-ups. The insidious course of R. equi disease and the difficulties in the isolation of the microorganism contribute to the delay in the diagnosis and to the high mortality rate of this opportunistic infection.

  16. Immunogenicity of an electron beam inactivated Rhodococcus equi vaccine in neonatal foals.

    PubMed

    Bordin, Angela I; Pillai, Suresh D; Brake, Courtney; Bagley, Kaytee B; Bourquin, Jessica R; Coleman, Michelle; Oliveira, Fabiano N; Mwangi, Waithaka; McMurray, David N; Love, Charles C; Felippe, Maria Julia B; Cohen, Noah D

    2014-01-01

    Rhodococcus equi is an important pathogen of foals that causes severe pneumonia. To date, there is no licensed vaccine effective against R. equi pneumonia of foals. The objectives of our study were to develop an electron beam (eBeam) inactivated vaccine against R. equi and evaluate its immunogenicity. A dose of eBeam irradiation that inactivated replication of R. equi while maintaining outer cell wall integrity was identified. Enteral administration of eBeam inactivated R. equi increased interferon-γ production by peripheral blood mononuclear cells in response to stimulation with virulent R. equi and generated naso-pharyngeal R. equi-specific IgA in newborn foals. Our results indicate that eBeam irradiated R. equi administered enterally produce cell-mediated and upper respiratory mucosal immune responses, in the face of passively transferred maternal antibodies, similar to those produced in response to enteral administration of live organisms (a strategy which previously has been documented to protect foals against intrabronchial infection with virulent R. equi). No evidence of adverse effects was noted among vaccinated foals.

  17. Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic acid chain length

    PubMed Central

    Sydor, Tobias; Bargen, Kristine; Hsu, Fong-Fu; Huth, Gitta; Holst, Otto; Wohlmann, Jens; Becken, Ulrike; Dykstra, Tobias; Söhl, Kristina; Lindner, Buko; Prescott, John F; Schaible, Ulrich E; Utermöhlen, Olaf; Haas, Albert

    2013-01-01

    Rhodococcus equi is a close relative of Mycobacterium spp. and a facultative intracellular pathogen which arrests phagosome maturation in macrophages before the late endocytic stage. We have screened a transposon mutant library of R. equi for mutants with decreased capability to prevent phagolysosome formation. This screen yielded a mutant in the gene for β-ketoacyl-(acyl carrier protein)-synthase A (KasA), a key enzyme of the long-chain mycolic acid synthesizing FAS-II system. The longest kasA mutant mycolic acid chains were 10 carbon units shorter than those of wild-type bacteria. Coating of non-pathogenic E. coli with purified wild-type trehalose dimycolate reduced phagolysosome formation substantially which was not the case with shorter kasA mutant-derived trehalose dimycolate. The mutant was moderately attenuated in macrophages and in a mouse infection model, but was fully cytotoxic.Whereas loss of KasA is lethal in mycobacteria, R. equi kasA mutant multiplication in broth was normal proving that long-chain mycolic acid compounds are not necessarily required for cellular integrity and viability of the bacteria that typically produce them. This study demonstrates a central role of mycolic acid chain length in diversion of trafficking by R. equi. PMID:23078612

  18. Nitric oxide-mediated intracellular growth restriction of pathogenic Rhodococcus equi can be prevented by iron.

    PubMed

    von Bargen, Kristine; Wohlmann, Jens; Taylor, Gregory Alan; Utermöhlen, Olaf; Haas, Albert

    2011-05-01

    Rhodococcus equi is an intracellular pathogen which causes pneumonia in young horses and in immunocompromised humans. R. equi arrests phagosome maturation in macrophages at a prephagolysosome stage and grows inside a privileged compartment. Here, we show that, in murine macrophages activated with gamma interferon and lipopolysaccharide, R. equi does not multiply but stays viable for at least 24 h. Whereas infection control of other intracellular pathogens by activated macrophages is executed by enhanced phagosome acidification or phagolysosome formation, by autophagy or by the interferon-inducible GTPase Irgm1, none of these mechanisms seems to control R. equi infection. Growth control by macrophage activation is fully mimicked by treatment of resting macrophages with nitric oxide donors, and inhibition of bacterial multiplication by either activation or nitric oxide donors is annihilated by cotreatment of infected macrophages with ferrous sulfate. Transcriptional analysis of the R. equi iron-regulated gene iupT demonstrates that intracellular R. equi encounters iron stress in activated, but not in resting, macrophages and that this stress is relieved by extracellular addition of ferrous sulfate. Our results suggest that nitric oxide is central to the restriction of bacterial access to iron in activated macrophages.

  19. Induction of proinflammatory cytokines in human lung epithelial cells during Rhodococcus equi infection.

    PubMed

    Remuzgo-Martínez, Sara; Pilares-Ortega, Lilian; Alvarez-Rodríguez, Lorena; Aranzamendi-Zaldunbide, Maitane; Padilla, Daniel; Icardo, Jose Manuel; Ramos-Vivas, Jose

    2013-08-01

    Rhodococcus equi is an opportunistic human pathogen associated with immunosuppressed people. While the interaction of R. equi with macrophages has been comprehensively studied, little is known about its interactions with non-phagocytic cells. Here, we characterized the entry process of this bacterium into human lung epithelial cells. The invasion is inhibited by nocodazole and wortmannin, suggesting that the phosphatidylinositol 3-kinase pathway and microtubule cytoskeleton are important for invasion. Pre-incubation of R. equi with a rabbit anti-R. equi polyclonal antiserum resulted in a dramatic reduction in invasion. Also, the invasion process as studied by immunofluorescence and scanning electron microscopy indicates that R. equi make initial contact with the microvilli of the A549 cells, and at the structural level, the entry process was observed to occur via a zipper-like mechanism. Infected lung epithelial cells upregulate the expression of cytokines IL-8 and IL-6 upon infection. The production of these pro-inflammatory cytokines was significantly enhanced in culture supernatants from cells infected with non-mucoid plasmid-less strains when compared with cells infected with mucoid strains. These results demonstrate that human airway epithelial cells produce pro-inflammatory mediators against R. equi isolates.

  20. Nitric Oxide-Mediated Intracellular Growth Restriction of Pathogenic Rhodococcus equi Can Be Prevented by Iron▿

    PubMed Central

    von Bargen, Kristine; Wohlmann, Jens; Taylor, Gregory Alan; Utermöhlen, Olaf; Haas, Albert

    2011-01-01

    Rhodococcus equi is an intracellular pathogen which causes pneumonia in young horses and in immunocompromised humans. R. equi arrests phagosome maturation in macrophages at a prephagolysosome stage and grows inside a privileged compartment. Here, we show that, in murine macrophages activated with gamma interferon and lipopolysaccharide, R. equi does not multiply but stays viable for at least 24 h. Whereas infection control of other intracellular pathogens by activated macrophages is executed by enhanced phagosome acidification or phagolysosome formation, by autophagy or by the interferon-inducible GTPase Irgm1, none of these mechanisms seems to control R. equi infection. Growth control by macrophage activation is fully mimicked by treatment of resting macrophages with nitric oxide donors, and inhibition of bacterial multiplication by either activation or nitric oxide donors is annihilated by cotreatment of infected macrophages with ferrous sulfate. Transcriptional analysis of the R. equi iron-regulated gene iupT demonstrates that intracellular R. equi encounters iron stress in activated, but not in resting, macrophages and that this stress is relieved by extracellular addition of ferrous sulfate. Our results suggest that nitric oxide is central to the restriction of bacterial access to iron in activated macrophages. PMID:21383050

  1. The sensor kinase MprB is required for Rhodococcus equi virulence.

    PubMed

    MacArthur, Iain; Parreira, Valeria R; Lepp, Dion; Mutharia, Lucy M; Vazquez-Boland, José A; Prescott, John F

    2011-01-10

    Rhodococcus equi is a soil bacterium and, like Mycobacterium tuberculosis, a member of the mycolata. Through possession of a virulence plasmid, it has the ability to infect the alveolar macrophages of foals, resulting in pyogranulomatous bronchopneumonia. The virulence plasmid has an orphan two-component system (TCS) regulatory gene, orf8, mutation of which completely attenuates virulence. This study attempted to find the cognate sensor kinase (SK) of orf8. Annotation of the R. equi strain 103 genome identified 23 TCSs encoded on the chromosome, which were used in a DNA microarray to compare TCS gene transcription in murine macrophage-like cells to growth in vitro. This identified six SKs as significantly up-regulated during growth in macrophages. Mutants of these SKs were constructed and their ability to persist in macrophages was determined with one SK, MprB, found to be required for intracellular survival. The attenuation of the mprB- mutant, and its complementation, was confirmed in a mouse virulence assay. In silico analysis of the R. equi genome sequence identified an MprA binding box motif homologous to that of M. tuberculosis, on mprA, pepD, sigB and sigE. The results of this study also show that R. equi responds to the macrophage environment differently from M. tuberculosis. MprB is the first SK identified as required for R. equi virulence and intracellular survival. Copyright © 2010 Elsevier B.V. All rights reserved.

  2. Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic acid chain length.

    PubMed

    Sydor, Tobias; von Bargen, Kristine; Hsu, Fong-Fu; Huth, Gitta; Holst, Otto; Wohlmann, Jens; Becken, Ulrike; Dykstra, Tobias; Söhl, Kristina; Lindner, Buko; Prescott, John F; Schaible, Ulrich E; Utermöhlen, Olaf; Haas, Albert

    2013-03-01

    Rhodococcus equi is a close relative of Mycobacterium spp. and a facultative intracellular pathogen which arrests phagosome maturation in macrophages before the late endocytic stage. We have screened a transposon mutant library of R. equi for mutants with decreased capability to prevent phagolysosome formation. This screen yielded a mutant in the gene for β-ketoacyl-(acyl carrier protein)-synthase A (KasA), a key enzyme of the long-chain mycolic acid synthesizing FAS-II system. The longest kasA mutant mycolic acid chains were 10 carbon units shorter than those of wild-type bacteria. Coating of non-pathogenic E. coli with purified wild-type trehalose dimycolate reduced phagolysosome formation substantially which was not the case with shorter kasA mutant-derived trehalose dimycolate. The mutant was moderately attenuated in macrophages and in a mouse infection model, but was fully cytotoxic.Whereas loss of KasA is lethal in mycobacteria, R. equi kasA mutant multiplication in broth was normal proving that long-chain mycolic acid compounds are not necessarily required for cellular integrity and viability of the bacteria that typically produce them. This study demonstrates a central role of mycolic acid chain length in diversion of trafficking by R. equi. © 2012 Blackwell Publishing Ltd.

  3. Development of a loop-mediated isothermal amplification method for detecting virulent Rhodococcus equi.

    PubMed

    Kinoshita, Yuta; Niwa, Hidekazu; Higuchi, Tohru; Katayama, Yoshinari

    2016-09-01

    Rhodococcus equi is the most important causative bacterium of severe pneumonia in foals. We report herein the development of a specific loop-mediated isothermal amplification (LAMP) assay, which targets a gene encoding vapA for detecting virulent R. equi The detection limit of the LAMP assay was 10(4) colony forming units (CFU)/mL, which was equal to 10 CFU/reaction. The clinical efficacy of the LAMP assay was compared with those of 2 published PCR-based methods: nested PCR and quantitative real-time (q)PCR. Agreements between bacterial culture, which is the gold standard for detection of R. equi, and each of the 3 molecular tests were measured by calculating a kappa coefficient. The kappa coefficients of the LAMP (0.760), nested PCR (0.583), and qPCR (0.888) indicated substantial agreement, moderate agreement, and almost perfect agreement, respectively. Although the clinical efficacy of LAMP was not the best among the 3 methods tested, LAMP could be more easily introduced into less well-equipped clinics because it does not require special equipment (such as a thermocycler) for gene amplification. Veterinary practitioners could diagnose R. equi pneumonia more quickly by using LAMP and could use the results to select an appropriate initial treatment. © 2016 The Author(s).

  4. Structure of the virulence-associated protein VapD from the intracellular pathogen Rhodococcus equi.

    PubMed

    Whittingham, Jean L; Blagova, Elena V; Finn, Ciaran E; Luo, Haixia; Miranda-CasoLuengo, Raúl; Turkenburg, Johan P; Leech, Andrew P; Walton, Paul H; Murzin, Alexey G; Meijer, Wim G; Wilkinson, Anthony J

    2014-08-01

    Rhodococcus equi is a multi-host pathogen that infects a range of animals as well as immune-compromised humans. Equine and porcine isolates harbour a virulence plasmid encoding a homologous family of virulence-associated proteins associated with the capacity of R. equi to divert the normal processes of endosomal maturation, enabling bacterial survival and proliferation in alveolar macrophages. To provide a basis for probing the function of the Vap proteins in virulence, the crystal structure of VapD was determined. VapD is a monomer as determined by multi-angle laser light scattering. The structure reveals an elliptical, compact eight-stranded β-barrel with a novel strand topology and pseudo-twofold symmetry, suggesting evolution from an ancestral dimer. Surface-associated octyl-β-D-glucoside molecules may provide clues to function. Circular-dichroism spectroscopic analysis suggests that the β-barrel structure is preceded by a natively disordered region at the N-terminus. Sequence comparisons indicate that the core folds of the other plasmid-encoded virulence-associated proteins from R. equi strains are similar to that of VapD. It is further shown that sequences encoding putative R. equi Vap-like proteins occur in diverse bacterial species. Finally, the functional implications of the structure are discussed in the light of the unique structural features of VapD and its partial structural similarity to other β-barrel proteins.

  5. Crystal structure and catalytic mechanism of chloromuconolactone dehalogenase ClcF from Rhodococcus opacus 1CP.

    PubMed

    Roth, Christian; Gröning, Janosch Alexander D; Kaschabek, Stefan Rudolf; Schlömann, Michael; Sträter, Norbert

    2013-04-01

    The actinobacterium Rhodococcus opacus 1CP possesses a so far unique variant of the modified 3-oxoadipate pathway for 3-chlorocatechol degradation. One important feature is the novel dehalogenase ClcF, which converts (4R,5S)-5-chloromuconolactone to E-dienelactone. ClcF is related to muconolactone isomerase (MLI, EC 5.3.3.4). The enzyme has a ferredoxin-type fold and forms a homodecamer of 52-symmetry, typical for the MLI family. The active site is formed by residues from two monomers. The complex structure of an E27A variant with bound substrate in conjunction with mutational studies indicate that E27 acts as the proton acceptor in a univalent single-base syn-dehydrohalogenation mechanism. Despite the evolutionary specialization of ClcF, the conserved active-site structures suggest that the proposed mechanism is representative for the MLI family. Furthermore, ClcF represents a novel type of dehalogenase based on an isomerase scaffold. © 2013 Blackwell Publishing Ltd.

  6. An Evaluation of Kinetic Models in the Biodesulfurization of Synthetic Oil by Rhodococcus erythropolis ATCC 4277.

    PubMed

    Maass, D; Mayer, D A; Moritz, D E; Oliveira, D; de Souza, A A Ulson; Souza, S M A Guelli

    2015-10-01

    Biodesulfurization is an eco-friendly technology applied in the removal of sulfur from fossil fuels. This technology is based on the use of microorganisms as biocatalysts to convert the recalcitrant sulfur compounds into others easily treatable, as sulfides. Despite it has been studied during the last decades, there are some unsolved questions, as per example the kinetic model which appropriately describes the biodesulfurization globally. In this work, different kinetic models were tested to a batch desulfurization process using dibenzothiophene (DBT) as a model compound, n-dodecane as organic solvent, and Rhodococcus erythropolis ATCC 4277 as biocatalyst. The models were solved by ODE45 function in the MATLAB. Monod model was capable to describe the biodesulfurization process predicting all experimental data with a very good fitting. The coefficients of determination achieved to organic phase concentrations of 20, 80, and 100 % (v/v) were 0.988, 0.995, and 0.990, respectively. R. erythropolis ATCC 4277 presented a good affinity with the substrate (DBT) since the coefficients of saturation obtained to reaction medium containing 20, 80, and 100 % (v/v) were 0.034, 0.07, and 0.116, respectively. This kinetic evaluation provides an improvement in the development of biodesulfurization technology because it showed that a simple model is capable to describe the throughout process.

  7. Comparative transcriptomics elucidates adaptive phenol tolerance and utilization in lipid-accumulating Rhodococcus opacus PD630

    DOE PAGES

    Yoneda, Aki; Henson, William R.; Goldner, Nicholas K.; ...

    2016-02-02

    Lignin-derived (e.g. phenolic) compounds can compromise the bioconversion of lignocellulosic biomass to fuels and chemicals due to their toxicity and recalcitrance. The lipid-accumulating bacterium Rhodococcus opacus PD630 has recently emerged as a promising microbial host for lignocellulose conversion to value-added products due to its natural ability to tolerate and utilize phenolics. To gain a better understanding of its phenolic tolerance and utilization mechanisms, we adaptively evolved R. opacus over 40 passages using phenol as its sole carbon source (up to 373% growth improvement over wild-type), and extensively characterized two strains from passages 33 and 40. The two adapted strains showedmore » higher phenol consumption rates (~20 mg/l/h) and ~2-fold higher lipid production from phenol than the wild-type strain.Whole-genome sequencing and comparative transcriptomics identified highly-upregulated degradation pathways and putative transporters for phenol in both adapted strains, highlighting the important linkage between mechanisms of regulated phenol uptake, utilization, and evolved tolerance. Our study shows that the R. opacus mutants are likely to use their transporters to import phenol rather than export them, suggesting a new aromatic tolerance mechanism. The identified tolerance genes and pathways are promising candidates for future metabolic engineering in R. opacus for improved lignin conversion to lipid-based products.« less

  8. Development of Chemical and Metabolite Sensors for Rhodococcus opacus PD630.

    PubMed

    DeLorenzo, Drew M; Henson, William R; Moon, Tae Seok

    2017-08-10

    Rhodococcus opacus PD630 is a nonmodel, Gram-positive bacterium that possesses desirable traits for biomass conversion, including consumption capabilities for lignocellulose-based sugars and toxic lignin-derived aromatic compounds, significant triacylglycerol accumulation, relatively rapid growth rate, and genetic tractability. However, few genetic elements have been directly characterized in R. opacus, limiting its application for lignocellulose bioconversion. Here, we report the characterization and development of genetic tools for tunable gene expression in R. opacus, including: (1) six fluorescent reporters for quantifying promoter output, (2) three chemically inducible promoters for variable gene expression, and (3) two classes of metabolite sensors derived from native R. opacus promoters that detect nitrogen levels or aromatic compounds. Using these tools, we also provide insights into native aromatic consumption pathways in R. opacus. Overall, this work expands the ability to control and characterize gene expression in R. opacus for future lignocellulose-based fuel and chemical production.

  9. Emulsification potential of a newly isolated biosurfactant-producing bacterium, Rhodococcus sp. strain TA6.

    PubMed

    Shavandi, Mahmoud; Mohebali, Ghasemali; Haddadi, Azam; Shakarami, Heidar; Nuhi, Ashrafossadat

    2011-02-01

    An indigenous biosurfactant producing bacterium, Rhodococcus sp. strain TA6 was isolated from Iranian oil contaminated soil using an efficient enrichment and screening method. During growth on sucrose and several hydrocarbon substrates as sole carbon source, the bacterium could produce biosurfactants. As a result of biosurfactant synthesis, the surface tension of the growth medium was reduced from 68mNm(-1) to values below 30mNm(-1). The biosurfactant was capable of forming stable emulsions with various hydrocarbons ranging from pentane to light motor oil. Preliminary chemical characterization revealed that the TA6 biosurfactant consisted of extracellular lipids and glycolipids. The biosurfactant was stable during exposure to high salinity (10% NaCl), elevated temperatures (120°C for 15min) and within a wide pH range (4.0-10.0). The culture broth was effective in recovering up to 70% of the residual oil from oil-saturated sand packs which indicates the potential value of the biosurfactant in enhanced oil recovery. Copyright © 2010 Elsevier B.V. All rights reserved.

  10. Optimal Production of a Rhodococcus erythropolis ATCC 4277 Biocatalyst for Biodesulfurization and Biodenitrogenation Applications.

    PubMed

    Todescato, Diego; Maass, Danielle; Mayer, Diego Alex; Vladimir Oliveira, J; de Oliveira, Débora; Ulson de Souza, Selene M A Guelli; Ulson de Souza, Antônio Augusto

    2017-05-20

    Rhodococcus sp. has a broad catabolic diversity and unique enzymatic capabilities, and it is able to adapt under extreme conditions. Thereby, the production of this remarkable bacterium has a great biotechnological and industrial importance. In this sense, we sought to improve the R. erythropolis ATCC 4277 growth through a central composite design, by varying the components of nutrient medium (glucose, malt extract, yeast extract, CaCO3), temperature, and agitation. It was found that the concentrations of glucose and malt extract are not statistically significant, being reduced of 4.0 and 10.0 g L(-1) to 2.0 and 5.0 g L(-1), respectively. The CaCO3 concentration and temperature were also diminished of 2.0 to 1.16 g L(-1)and 28 to 23.7 °C, respectively. Optimal growth conditions provided a 240% increase in final biomass concentration, an increment in specific growth rate, and a growth yield coefficient about five times greater. Application of the optimal conditions in biodesulfurization and biodenitrogenation processes showed that desulfurization capability is not associated with optimal growth conditions; however, it was achieved a 47% of nitrogen removal in the assay containing 10% (w/w) of heavy gas oil. Graphical Abstract ᅟ.

  11. Microbial degradation of four crude oil by biosurfactant producing strain Rhodococcus sp.

    PubMed

    Pi, Yongrui; Chen, Bing; Bao, Mutai; Fan, Fuqiang; Cai, Qinhong; Ze, Lv; Zhang, Baiyu

    2017-05-01

    Rhodococcus erythropolis M-25, one of the representative biosurfactant producers, performed effectively during the biodegradation of four crude oil. The microbial degradation efficiency is positively relevant to the API of the crude oil. The chemical dispersant Corexit 9500A did not enhance the biodegradation of the petroleum hydrocarbons during the experimental period. 70.7% of the N-4 oil was degraded after 30days, while in the Corexit 9500A plus sample the biodegradation removal was 42.8%. The Corexit-derived compounds were metabolized by M-25 at the same time of the petroleum hydrocarbons biodegrading. Neither biodegradation nor chemical dispersion process has almost no effect on the biomarker (m/z=231). The saturated methyl-branched fatty acids increased from 37.3%, to 49.4%, when M-25 was exposed with the N-4 crude oil. Similarly, the saturated methyl-branched fatty acids in the membrane of N3-2P increased from 20.25% to 44.1%, when exposed it with the N-4 crude oil.

  12. Crystallization and preliminary characterization of chloromuconolactone dehalogenase from Rhodococcus opacus 1CP.

    PubMed

    Roth, Christian; Kaschabek, Stefan R; Gröning, Janosch A D; Handrek, Thomas; Schlömann, Michael; Sträter, Norbert

    2012-05-01

    Chloroaromatic compounds are often very persistent environmental pollutants. Nevertheless, numerous bacteria are able to metabolize these compounds and to utilize them as sole energy and carbon sources. Rhodococcus opacus 1CP is able to degrade several chloroaromatic compounds, some of them via a variation of the 3-chlorocatechol branch of the modified ortho-cleavage pathway. This branch in R. opacus differs from that in Proteobacteria in the inability of the chloromuconate cycloisomerase to dehalogenate. Instead, a unique enzyme designated as chloromuconolactone dehalogenase (ClcF) is recruited. ClcF dehalogenates 5-chloromuconolactone to cis-dienelactone and shows a high similarity to muconolactone isomerases (EC 5.3.3.4). However, unlike the latter enzymes, it is unable to catalyse the isomerization of muconolactone to 3-oxoadipate enollactone. In order to characterize the catalytic mechanism of this unusual dehalogenase, the enzyme was crystallized and subjected to X-ray structural analysis. Data sets to up to 1.65 Å resolution were collected from two different crystal forms using synchrotron radiation. Crystal form I (space group P2(1)) contained 40 subunits in the asymmetric unit, whereas ten subunits were present in crystal form II (space group P2(1)2(1)2(1)). The self-rotation function revealed the orientations of the molecular symmetry axes of the homodecamer of 52 symmetry.

  13. Mining the genome of Rhodococcus fascians, a plant growth-promoting bacterium gone astray.

    PubMed

    Francis, Isolde M; Stes, Elisabeth; Zhang, Yucheng; Rangel, Diana; Audenaert, Kris; Vereecke, Danny

    2016-09-25

    Rhodococcus fascians is a phytopathogenic Gram-positive Actinomycete with a very broad host range encompassing especially dicotyledonous herbaceous perennials, but also some monocots, such as the Liliaceae and, recently, the woody crop pistachio. The pathogenicity of R. fascians strain D188 is known to be encoded by the linear plasmid pFiD188 and to be dictated by its capacity to produce a mixture of cytokinins. Here, we show that D188-5, the nonpathogenic plasmid-free derivative of the wild-type strain D188 actually has a plant growth-promoting effect. With the availability of the genome sequence of R. fascians, the chromosome of strain D188 was mined for putative plant growth-promoting functions and the functionality of some of these activities was tested. This analysis together with previous results suggests that the plant growth-promoting activity of R. fascians is due to production of plant growth modulators, such as auxin and cytokinin, combined with degradation of ethylene through 1-amino-cyclopropane-1-carboxylic acid deaminase. Moreover, R. fascians has several functions that could contribute to efficient colonization and competitiveness, but there is little evidence for a strong impact on plant nutrition. Possibly, the plant growth promotion encoded by the D188 chromosome is imperative for the epiphytic phase of the life cycle of R. fascians and prepares the plant to host the bacteria, thus ensuring proper continuation into the pathogenic phase. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Biodesulfurization of water-soluble coal-derived material by Rhodococcus rhodochrous IGTS8

    SciTech Connect

    Kilbane, J.J. II; Jackowski, K.

    1991-12-31

    Rhodococcus rhodochrous IGTS8 was previously isolated because of its ability to use coal as its sole source of sulfur for growth. Subsequent growth studies have revealed that IGTS8 is capable of using a variety of organosulfur compounds as sources of sulfur but not carbon. In this paper, the ability of IGTS8 to selectively remove organic sulfur from water-soluble coal-derived material is investigated. The microbial removal of organic sulfur from coal requires microorganisms capable of cleaving carbonsulfur bonds and the accessibility of these bonds to microorganisms. The use of water-soluble coal-derived material effectively overcomes the problem of accessibility and allows the ability of microorganisms to cleave carbonsulfur bonds present in coal-derived material to be assessed directly. Three coals, two coal solubilization procedures, and two methods of biodesulfurization were examined. The results of these experiments reveal that the microbial removal of significant amounts of organic sulfur from watersoluble coal-derived material with treatment times as brief as 24 hours is possible. Moreover, the carbon content and calorific value of biotreated products are largely unaffected. Biotreatment does, however, result in increases in the hydrogen and nitrogen content and a decreased oxygen content of the coal-derived material. The aqueous supernatant obtained from biodesulfurization experiments does not contain sulfate, sulfite, or other forms of soluble sulfur at increased concentrations in comparison with control samples. Sulfur removed from water-soluble coal-derived material appears to be incorporated into biomass.

  15. Biodegradation of buprofezin by Rhodococcus sp. strain YL-1 isolated from rice field soil.

    PubMed

    Li, Chao; Zhang, Ji; Wu, Zhi-Guo; Cao, Li; Yan, Xin; Li, Shun-Peng

    2012-03-14

    A buprofezin-degrading bacterium, YL-1, was isolated from rice field soil. YL-1 was identified as Rhodococcus sp. on the basis of the comparative analysis of 16S rDNA sequences. The strain could use buprofezin as the sole source of carbon and nitrogen for growth and was able to degrade 92.4% of 50 mg L(-1) buprofezin within 48 h in liquid culture. During the degradation of buprofezin, four possible metabolites, 2-tert-butylimino-3-isopropyl-1,3,5-thiadiazinan-4-one, N-tert-butyl-thioformimidic acid formylaminomethyl ester, 2-isothiocyanato-2-methyl-propane, and 2-isothiocyanato-propane, were identified using gas chromatography-mass spectrometry (GC-MS) analysis. The catechol 2,3-dioxygenase activity was strongly induced during the degradation of buprofezin. A novel microbial biodegradation pathway for buprofezin was proposed on the basis of these metabolites. The inoculation of soils treated with buprofezin with strain YL-1 resulted in a higher degradation rate than that observed in noninoculated soils, indicating that strain YL-1 has the potential to be used in the bioremediation of buprofezin-contaminated environments.

  16. Biodegradation of pharmaceuticals by Rhodococcus rhodochrous and Aspergillus niger by co-metabolism.

    PubMed

    Gauthier, Hervé; Yargeau, Viviane; Cooper, David G

    2010-03-01

    This work investigated the possible fate of pharmaceuticals in the environment that are known to be resistant to biodegradation. A co-metabolism approach, adding a readily degradable carbon source, was used to study the biodegradation of some pharmaceuticals. The pharmaceuticals selected were all known to be micro pollutants and frequently used by humans. The microorganisms used primarily were Rhodococcus rhodochrous, known to co-metabolize difficult to degrade hydrocarbons and Aspergillus niger. Because of the long periods of time required for the degradation experiments after growth had reached the stationary phase, it was found to be necessary to correct for water loss from the media. Co-metabolism of carbamazepine, sulfamethizole and sulfamethoxazole was observed and as much as 20% of these compounds could be removed. Small amounts of stable metabolites were observed during the degradation of some of these drugs and these were different from the metabolites obtained from abiotic degradation. A metabolite arising from the biodegradation of sulfamethoxazole by R.rhodochrous was identified.

  17. High-level expression in Corynebacterium glutamicum of nitrile hydratase from Rhodococcus rhodochrous for acrylamide production.

    PubMed

    Kang, Mi-Suk; Han, Sang-Soo; Kim, Mi-Young; Kim, Bu-Youn; Huh, Jong-Pil; Kim, Hak-Sung; Lee, Jin-Ho

    2014-05-01

    The nhhBAG gene of Rhodococcus rhodochrous M33 that encodes nitrile hydratase (NHase), converting acrylonitrile into acrylamide, was cloned and expressed in Corynebacterium glutamicum under the control of an ilvC promoter. The specific enzyme activity in recombinant C. glutamicum cells was about 13.6 μmol/min/mg dry cell weight (DCW). To overexpress the NHase, five types of plasmid variants were constructed by introducing mutations into 80 nucleotides near the translational initiation region (TIR) of nhhB. Of them, pNBM4 with seven mutations showed the highest NHase activity, exhibiting higher expression levels of NhhB and NhhA than wild-type pNBW33, mainly owing to decreased secondary-structure stability and an introduction of a conserved Shine-Dalgarno sequence in the translational initiation region. In a fed-batch culture of recombinant Corynebacterium cells harboring pNBM4, the cell density reached 53.4 g DCW/L within 18 h, and the specific and total enzyme activities were estimated to be 37.3 μmol/min/mg DCW and 1,992 μmol/min/mL, respectively. The use of recombinant Corynebacterium cells for the production of acrylamide from acrylonitrile resulted in a conversion yield of 93 % and a final acrylamide concentration of 42.5 % within 6 h when the total amount of fed acrylonitrile was 456 g.

  18. Metabolic responses of Rhodococcus erythropolis PR4 grown on diesel oil and various hydrocarbons.

    PubMed

    Laczi, Krisztián; Kis, Ágnes; Horváth, Balázs; Maróti, Gergely; Hegedüs, Botond; Perei, Katalin; Rákhely, Gábor

    2015-11-01

    Rhodococcus erythropolis PR4 is able to degrade diesel oil, normal-, iso- and cycloparaffins and aromatic compounds. The complete DNA content of the strain was previously sequenced and numerous oxygenase genes were identified. In order to identify the key elements participating in biodegradation of various hydrocarbons, we performed a comparative whole transcriptome analysis of cells grown on hexadecane, diesel oil and acetate. The transcriptomic data for the most prominent genes were validated by RT-qPCR. The expression of two genes coding for alkane-1-monooxygenase enzymes was highly upregulated in the presence of hydrocarbon substrates. The transcription of eight phylogenetically diverse cytochrome P450 (cyp) genes was upregulated in the presence of diesel oil. The transcript levels of various oxygenase genes were determined in cells grown in an artificial mixture, containing hexadecane, cycloparaffin and aromatic compounds and six cyp genes were induced by this hydrocarbon mixture. Five of them were not upregulated by linear and branched hydrocarbons. The expression of fatty acid synthase I genes was downregulated by hydrocarbon substrates, indicating the utilization of external alkanes for fatty acid synthesis. Moreover, the transcription of genes involved in siderophore synthesis, iron transport and exopolysaccharide biosynthesis was also upregulated, indicating their important role in hydrocarbon metabolism. Based on the results, complex metabolic response profiles were established for cells grown on various hydrocarbons. Our results represent a functional annotation of a rhodococcal genome, provide deeper insight into molecular events in diesel/hydrocarbon utilization and suggest novel target genes for environmental monitoring projects.

  19. [Isolation, identification and degradation characteristics of a quinoline-degrading bacterium Rhodococcus sp QL2].

    PubMed

    Zhu, Shun-ni; Liu, Dong-qi; Fan, Li; Ni, Jin-ren

    2008-02-01

    A quinoline-degrading bacterium QL2, which utilizes quinoline as sole source of carbon, nitrogen and energy, was isolated from activated sludge in a coke-plant wastewater biological treatment system. According to the morphological characteristics, physiological and biochemical characteristics, and sequence analysis of 16S rRNA, the strain was identified as Rhodococcus sp.. The optimal temperature, initial pH, and shaker rotary speed for strain QL2 utilizing quinoline are 35-42 degrees C, pH 8-9, and 150 r/min, respectively. Extra nitrogen sources stimulate the isolate growth on quinoline, and inorganic nitrogen better than organic nitrogen, NH4+ -N better than NO3(-) -N. The degradation reaction of quinoline by strain QL2 can be described with zero order kinetic equation within the initial quinoline concentrations of 60-680 mg/L. When the initial concentration was 150 mg/L, quinoline was degraded completely in 8 hours and TOC removal efficiency was 70% in 14 hours. This bacterium produced pigmented compounds, and ring nitrogen was released into the growth medium as ammonium. The main intermediate in the degradation pathway was 2-hydroxyquinoline by the analysis of HPLC and GC/MS. With a broad range of substrate utilization, the strain can degrade phenol, naphthalene, pyridine, and some other kinds of aromatic compounds.

  20. Benzoate degradation by Rhodococcus opacus 1CP after dormancy: Characterization of dioxygenases involved in the process.

    PubMed

    Solyanikova, Inna P; Emelyanova, Elena V; Borzova, Oksana V; Golovleva, Ludmila A

    2016-01-01

    The process of benzoate degradation by strain Rhodococcus opacus 1CP after a five-year dormancy was investigated and its peculiarities were revealed. The strain was shown to be capable of growth on benzoate at a concentration of up to 10 g L(-1). The substrate specificity of benzoate dioxygenase (BDO) during the culture growth on a medium with a low (200-250 mg L(-1)) and high (4 g L(-1)) concentration of benzoate was assessed. BDO of R. opacus 1CP was shown to be an extremely narrow specificity enzyme. Out of 31 substituted benzoates, only with one, 3-chlorobenzoate, its activity was higher than 9% of that of benzoate. Two dioxygenases, catechol 1,2-dioxygenase (Cat 1,2-DO) and protocatechuate 3,4-dioxygenase (PCA 3,4-DO), were identified in a cell-free extract, purified and characterized. The substrate specificity of Cat 1,2-DO isolated from cells of strain 1CP after the dormancy was found to differ significantly from that of Cat 1,2-DO isolated earlier from cells of this strain grown on benzoate. By its substrate specificity, the described Cat 1,2-DO was close to the Cat 1,2-DO from strain 1CP grown on 4-methylbenzoate. Neither activity nor inhibition by protocatechuate was observed during the reaction of Cat 1,2-DO with catechol, and catechol had no inhibitory effect on the reaction of PCA 3,4-DO with protocatechuate.

  1. Deep Desulfurization of Diesel Oil and Crude Oils by a Newly Isolated Rhodococcus erythropolis Strain

    PubMed Central

    Yu, Bo; Xu, Ping; Shi, Quan; Ma, Cuiqing

    2006-01-01

    The soil-isolated strain XP was identified as Rhodococcus erythropolis. R. erythropolis XP could efficiently desulfurize benzonaphthothiophene, a complicated model sulfur compound that exists in crude oil. The desulfurization product of benzonaphthothiophene was identified as α-hydroxy-β-phenyl-naphthalene. Resting cells could desulfurize diesel oil (total organic sulfur, 259 ppm) after hydrodesulfurization. The sulfur content of diesel oil was reduced by 94.5% by using the resting cell biocatalyst for 24 h at 30°C. Biodesulfurization of crude oils was also investigated. After 72 h of treatment at 30°C, 62.3% of the total sulfur content in Fushun crude oil (initial total sulfur content, 3,210 ppm) and 47.2% of that in Sudanese crude oil (initial total sulfur, 1,237 ppm) were removed. Gas chromatography with pulsed-flame photometric detector analysis was used to evaluate the effect of R. erythropolis XP treatment on the sulfur content in Fushun crude oil, and it was shown that most organic sulfur compounds were eliminated after biodesulfurization. PMID:16391024

  2. Induction of Viable but Nonculturable State in Rhodococcus and Transcriptome Analysis Using RNA-seq

    PubMed Central

    Su, Xiaomei; Guo, Li; Ding, Linxian; Qu, Kun; Shen, Chaofeng

    2016-01-01

    Viable but nonculturable (VBNC) bacteria, which maintain the viability with loss of culturability, universally exist in contaminated and non-contaminated environments. In this study, two strains, Rhodococcus sp. TG13 and TN3, which were isolated from PCB-contaminated sediment and non-contaminated sediment respectively, were investigated under low temperature and oligotrophic conditions. The results indicated that the two strains TG13 and TN3 could enter into the VBNC state with different incubation times, and could recover culturability by reversal of unfavourable factors and addition of resuscitation-promoting factor (Rpf), respectively. Furthermore, the gene expression variations in the VBNC response were clarified by Illumina high throughput RNA-sequencing. Genome-wide transcriptional analysis demonstrated that up-regulated genes in the VBNC cells of the strain TG13 related to protein modification, ATP accumulation and RNA polymerase, while all differentially expressed genes (DEGs) in the VBNC cells of the strain TN3 were down-regulated. However, the down-regulated genes in both the two strains mainly encoded NADH dehydrogenase subunit, catalase, oxidoreductase, which further verified that cold-induced loss of ability to defend oxidative stress may play an important role in induction of the VBNC state. This study further verified that the molecular mechanisms underlying the VBNC state varied with various bacterial species. Study on the VBNC state of non-pathogenic bacteria will provide new insights into the limitation of environmental micro-bioremediation and the cultivation of unculturable species. PMID:26808070

  3. Enzymatic degradation of aliphatic nitriles by Rhodococcus rhodochrous BX2, a versatile nitrile-degrading bacterium.

    PubMed

    Fang, Shumei; An, Xuejiao; Liu, Hongyuan; Cheng, Yi; Hou, Ning; Feng, Lu; Huang, Xinning; Li, Chunyan

    2015-06-01

    Nitriles are common environmental pollutants, and their removal has attracted increasing attention. Microbial degradation is considered to be the most acceptable method for removal. In this work, we investigated the biodegradation of three aliphatic nitriles (acetonitrile, acrylonitrile and crotononitrile) by Rhodococcus rhodochrous BX2 and the expression of their corresponding metabolic enzymes. This organism can utilize all three aliphatic nitriles as sole carbon and nitrogen sources, resulting in the complete degradation of these compounds. The degradation kinetics were described using a first-order model. The degradation efficiency was ranked according to t1/2 as follows: acetonitrile>trans-crotononitrile>acrylonitrile>cis-crotononitrile. Only ammonia accumulated following the three nitriles degradation, while amides and carboxylic acids were transient and disappeared by the end of the assay. mRNA expression and enzyme activity indicated that the tested aliphatic nitriles were degraded via both the inducible NHase/amidase and the constitutive nitrilase pathways, with the former most likely preferred. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Mathematic Modeling for Optimum Conditions on Aflatoxin B1 Degradation by the Aerobic Bacterium Rhodococcus erythropolis

    PubMed Central

    Kong, Qing; Zhai, Cuiping; Guan, Bin; Li, Chunjuan; Shan, Shihua; Yu, Jiujiang

    2012-01-01

    Response surface methodology was employed to optimize the degradation conditions of AFB1 by Rhodococcus erythropolis in liquid culture. The most important factors that influence the degradation, as identified by a two-level Plackett-Burman design with six variables, were temperature, pH, liquid volume, inoculum size, agitation speed and incubation time. Central composite design (CCD) and response surface analysis were used to further investigate the interactions between these variables and to optimize the degradation efficiency of R. erythropolis based on a second-order model. The results demonstrated that the optimal parameters were: temperature, 23.2 °C; pH, 7.17; liquid volume, 24.6 mL in 100-mL flask; inoculum size, 10%; agitation speed, 180 rpm; and incubation time, 81.9 h. Under these conditions, the degradation efficiency of R. erythropolis could reach 95.8% in liquid culture, which was increased by about three times as compared to non-optimized conditions. The result by mathematic modeling has great potential for aflatoxin removal in industrial fermentation such as in food processing and ethanol production. PMID:23202311

  5. pFiD188, the linear virulence plasmid of Rhodococcus fascians D188.

    PubMed

    Francis, Isolde; De Keyser, Annick; De Backer, Philippe; Simón-Mateo, Carmen; Kalkus, Jutta; Pertry, Ine; Ardiles-Diaz, Wilson; De Rycke, Riet; Vandeputte, Olivier M; El Jaziri, Mondher; Holsters, Marcelle; Vereecke, Danny

    2012-05-01

    Rhodococcus fascians is currently the only phytopathogen of which the virulence genes occur on a linear plasmid. To get insight into the origin of this replicon and into the virulence strategy of this broad-spectrum phytopathogen, the sequence of the linear plasmid of strain D188, pFiD188, was determined. Analysis of the 198,917 bp revealed four syntenic regions with linear plasmids of R. erythropolis, R. jostii, and R. opacus, suggesting a common origin of these replicons. Mutational analysis of pFi_086 and pFi_102, similar to cutinases and type IV peptidases, respectively, showed that conserved region R2 was involved in plasmid dispersal and pointed toward a novel function for actinobacterial cutinases in conjugation. Additionally, pFiD188 had three regions that were unique for R. fascians. Functional analysis of the stk and nrp loci of regions U2 and U3, respectively, indicated that their role in symptom development was limited compared with that of the previously identified fas, att, and hyp virulence loci situated in region U1. Thus, pFiD188 is a typical rhodococcal linear plasmid with a composite structure that encodes core functions involved in plasmid maintenance and accessory functions, some possibly acquired through horizontal gene transfer, implicated in virulence and the interaction with the host.

  6. Rhodococcus fascians impacts plant development through the dynamic fas-mediated production of a cytokinin mix.

    PubMed

    Pertry, Ine; Václavíková, Katerina; Gemrotová, Markéta; Spíchal, Lukás; Galuszka, Petr; Depuydt, Stephen; Temmerman, Wim; Stes, Elisabeth; De Keyser, Annick; Riefler, Michael; Biondi, Stefania; Novák, Ondrej; Schmülling, Thomas; Strnad, Miroslav; Tarkowski, Petr; Holsters, Marcelle; Vereecke, Danny

    2010-09-01

    The phytopathogenic actinomycete Rhodococcus fascians D188 relies mainly on the linear plasmid-encoded fas operon for its virulence. The bacteria secrete six cytokinin bases that synergistically redirect the developmental program of the plant to stimulate proliferation of young shoot tissue, thus establishing a leafy gall as a niche. A yeast-based cytokinin bioassay combined with cytokinin profiling of bacterial mutants revealed that the fas operon is essential for the enhanced production of isopentenyladenine, trans-zeatin, cis-zeatin, and the 2-methylthio derivatives of the zeatins. Cytokinin metabolite data and the demonstration of the enzymatic activities of FasD (isopentenyltransferase), FasE (cytokinin oxidase/dehydrogenase), and FasF (phosphoribohydrolase) led us to propose a pathway for the production of the cytokinin spectrum. Further evaluation of the pathogenicity of different fas mutants and of fas gene expression and cytokinin signal transduction upon infection implied that the secretion of the cytokinin mix is a highly dynamic process, with the consecutive production of a tom initiation wave followed by a maintenance flow.

  7. Transformation of Rhodococcus fascians by High-Voltage Electroporation and Development of R. fascians Cloning Vectors.

    PubMed

    Desomer, J; Dhaese, P; Montagu, M V

    1990-09-01

    The analysis of the virulence determinants of phytopathogenic Rhodococcus fascians has been hampered by the lack of a system for introducing exogenous DNA. We investigated the possibility of genetic transformation of R. fascians by high-voltage electroporation of intact bacterial cells in the presence of plasmid DNA. Electrotransformation in R. fascians D188 resulted in transformation frequencies ranging from 10/mug of DNA to 10/mug of DNA, depending on the DNA concentration. The effects of different electrical parameters and composition of electroporation medium on transformation efficiency are presented. By this transformation method, a cloning vector (pRF28) for R. fascians based on an indigenous 160-kilobase (chloramphenicol and cadmium resistance-encoding) plasmid pRF2 from strain NCPPB 1675 was developed. The origin of replication and the chloramphenicol resistance gene on pRF28 were used to construct cloning vectors that are capable of replication in R. fascians and Escherichia coli. The electroporation method presented was efficient enough to allow detection of the rare integration of replication-deficient pRF28 derivatives in the R. fascians D188 genome via either homologous or illegitimate recombination.

  8. Eternal youth, the fate of developing Arabidopsis leaves upon Rhodococcus fascians infection.

    PubMed

    Depuydt, Stephen; De Veylder, Lieven; Holsters, Marcelle; Vereecke, Danny

    2009-03-01

    The phytopathogenic actinomycete Rhodococcus fascians induces neoplastic shooty outgrowths on infected hosts. Upon R. fascians infection of Arabidopsis (Arabidopsis thaliana), leaves are formed with small narrow lamina and serrated margins. These symptomatic leaves exhibit reduced tissue differentiation, display more but smaller cells that do not endoreduplicate, and accumulate in the G1 phase of the cell cycle. Together, these features imply that leaf growth occurs primarily through mitotic cell division and not via cell expansion. Molecular analysis revealed that cell cycle gene expression is activated continuously throughout symptomatic leaf development, ensuring persistent mitotic cycling and inhibition of cell cycle exit. The transition at the two major cell cycle checkpoints is stimulated as a direct consequence of the R. fascians signals. The extremely reduced phenotypical response of a cyclind3;1-3 triple knockout mutant indicates that the D-type cyclin/retinoblastoma/E2F transcription factor pathway, as a major mediator of cell growth and cell cycle progression, plays a key role in symptom development and is instrumental for the sustained G1-to-S and G2-to-M transitions during symptomatic leaf growth.

  9. An integrated genomics approach to define niche establishment by Rhodococcus fascians.

    PubMed

    Depuydt, Stephen; Trenkamp, Sandra; Fernie, Alisdair R; Elftieh, Samira; Renou, Jean-Pierre; Vuylsteke, Marnik; Holsters, Marcelle; Vereecke, Danny

    2009-03-01

    Rhodococcus fascians is a Gram-positive phytopathogen that induces shooty hyperplasia on its hosts through the secretion of cytokinins. Global transcriptomics using microarrays combined with profiling of primary metabolites on infected Arabidopsis (Arabidopsis thaliana) plants revealed that this actinomycete modulated pathways to convert its host into a niche. The transcript data demonstrated that R. fascians leaves a very characteristic mark on Arabidopsis with a pronounced cytokinin response illustrated by the activation of cytokinin perception, signal transduction, and homeostasis. The microarray data further suggested active suppression of an oxidative burst during the R. fascians pathology, and comparison with publicly available transcript data sets implied a central role for auxin in the prevention of plant defense activation. Gene Ontology categorization of the differentially expressed genes hinted at a significant impact of infection on the primary metabolism of the host, which was confirmed by subsequent metabolite profiling. The much higher levels of sugars and amino acids in infected plants are presumably accessed by the bacteria as carbon and nitrogen sources to support epiphytic and endophytic colonization. Hexoses, accumulating from a significantly increased invertase activity, possibly inhibited the expression of photosynthesis genes and photosynthetic activity in infected leaves. Altogether, these changes are indicative of sink development in symptomatic tissues. The metabolomics data furthermore point to the possible occurrence of secondary signaling during the interaction, which might contribute to symptom development. These data are placed in the context of regulation of bacterial virulence gene expression, suppression of defense, infection phenotype, and niche establishment.

  10. Modulation of the hormone setting by Rhodococcus fascians results in ectopic KNOX activation in Arabidopsis.

    PubMed

    Depuydt, Stephen; Dolezal, Karel; Van Lijsebettens, Mieke; Moritz, Thomas; Holsters, Marcelle; Vereecke, Danny

    2008-03-01

    The biotrophic actinomycete Rhodococcus fascians has a profound impact on plant development and a common aspect of the symptomatology is the deformation of infected leaves. In Arabidopsis (Arabidopsis thaliana), the serrated leaf margins formed upon infection resemble the leaf phenotype of transgenic plants with ectopic expression of KNOTTED-like homeobox (KNOX) genes. Through transcript profiling, we demonstrate that class-I KNOX genes are transcribed in symptomatic leaves. Functional analysis revealed that BREVIPEDICELLUS/KNOTTED-LIKE1 and mainly SHOOT MERISTEMLESS were essential for the observed leaf dissection. However, these results also positioned the KNOX genes downstream in the signaling cascade triggered by R. fascians infection. The much faster activation of ARABIDOPSIS RESPONSE REGULATOR5 and the establishment of homeostatic and feedback mechanisms to control cytokinin (CK) levels support the overrepresentation of this hormone in infected plants due to the secretion by the pathogen, thereby placing the CK response high up in the cascade. Hormone measurements show a net decrease of tested CKs, indicating either that secretion by the bacterium and degradation by the plant are in balance, or, as suggested by the strong reaction of 35S:CKX plants, that other CKs are at play. At early time points of the interaction, activation of gibberellin 2-oxidase presumably installs a local hormonal setting favorable for meristematic activity that provokes leaf serrations. The results are discussed in the context of symptom development, evasion of plant defense, and the establishment of a specific niche by R. fascians.

  11. The Rhodococcus fascians-plant interaction: morphological traits and biotechnological applications.

    PubMed

    Vereecke, D; Burssens, S; Simón-Mateo, C; Inzé, D; Van Montagu, M; Goethals, K; Jaziri, M

    2000-01-01

    Rhodococcus fascians is a Gram-positive bacterium that infects dicotyledonous and monocotyledonous plants, leading to an alteration in the normal growth process of the host. The disease results from the modulation of the plant hormone balances, and cytokinins are thought to play an important role in the induction of symptoms. Generally, on the aerial parts of the plants, existing meristems were found to be most sensitive to the action of R. fascians, but, depending on the infection procedure, differentiated tissues as well gave rise to shoots. Similarly, in roots not only actively dividing cells, but also cells with a high competence to divide were strongly affected by R. fascians. The observed symptoms, together with the determined hormone levels in infected plant tissue, suggest that auxins and molecules of bacterial origin are also involved in leafy gall formation. The complexity of symptom development is furthermore illustrated by the necessary and continuous presence of the bacteria for symptom persistence. Indeed, elimination of the bacteria from a leafy gall results in the further development of the multiple embryonic buds of which it consists. This interesting characteristic offers novel biotechnological applications: a leafy gall can be used for germplasm storage and for plant propagation. The presented procedure proves to be routinely applicable to a very wide range of plants, encompassing several recalcitrant species.

  12. De novo cortical cell division triggered by the phytopathogen Rhodococcus fascians in tobacco.

    PubMed

    de O Manes, C L; Van Montagu, M; Prinsen, E; Goethals, K; Holsters, M

    2001-02-01

    Plant growth, development, and morphology can be affected by several environmental stimuli and by specific interactions with phytopathogens. In many cases, plants respond to pathogenic stimuli by adapting their hormone levels. Here, the interaction between the phytopathogen Rhodococcus fascians and one of its host plants, tobacco, was analyzed phenotypically and molecularly. To elucidate the basis of the cell division modulation and shoot primordia initiation caused by R. fascians, tobacco plants were infected at leaf axils and shoot apices. Adventitious meristems that gave rise to multiple-shoot primordia (leafy galls) were formed. The use of a transgenic line carrying the mitotic CycB1 promoter fused to the reporter gene coding for beta-glucuronidase from Escherichia coli (uidA), revealed that stem cortical cells were stimulated to divide in an initial phase of the leafy gall ontogenesis. Local cytokinin and auxin levels throughout the infection process as well as modulation of expression of the cell cycle regulator gene Nicta;CycD3;2 are discussed.

  13. Plant-derived auxin plays an accessory role in symptom development upon Rhodococcus fascians infection.

    PubMed

    Stes, Elisabeth; Prinsen, Els; Holsters, Marcelle; Vereecke, Danny

    2012-05-01

    The biotrophic phytopathogen Rhodococcus fascians has a profound impact on plant development, mainly through its principal virulence factors, a mix of synergistically acting cytokinins that induce shoot formation. Expression profiling of marker genes for several auxin biosynthesis routes and mutant analysis demonstrated that the bacterial cytokinins stimulate the auxin biosynthesis of plants via specific targeting of the indole-3-pyruvic acid (IPA) pathway, resulting in enhanced auxin signaling in infected tissues. The double mutant tryptophan aminotransferase 1-1 tryptophan aminotransferase related 2-1 (taa1-1 tar2-1) of Arabidopsis (Arabidopsis thaliana), in which the IPA pathway is defective, displayed a decreased responsiveness towards R. fascians infection, although bacterial colonization and virulence gene expression were not impaired. These observations implied that plant-derived auxin was employed to reinforce symptom formation. Furthermore, the increased auxin production and, possibly, the accumulating bacterial cytokinins in infected plants modified the polar auxin transport so that new auxin maxima were repetitively established and distributed, a process that is imperative for symptom onset and maintenance. Based on these findings, we extend our model of the mode of action of bacterial and plant signals during the interaction between R. fascians and Arabidopsis.

  14. Transformation of Rhodococcus fascians by high-voltage electroporation and development of R. fascians cloning vectors

    SciTech Connect

    Desomer, J.; Dhaese, P.; Montagu, M.V. )

    1990-09-01

    The analysis of the virulence determinants of phytopathogenic Rhodococcus fascians has been hampered by the lack of a system for introducing exogenous DNA. We investigated the possibility of genetic transformation of R. fascians by high-voltage electroporation of intact bacterial cells in the presence of plasmid DNA. Electrotransformation in R. fascians D188 resulted in transformation frequencies ranging from 10{sup 5}/{mu}g of DNA to 10{sup 7}/{mu}g of DNA, depending on the DNA concentration. The effects of different electrical parameters and composition of electroporation medium on transformation efficiency are present. By this transformation method, a cloning vector (pRF28) for R. fascians based on an indigenous 160-kilobase (chloramphenicol and cadmium resistance-encoding) plasmid pRF2 from strain NCPPB 1675 was developed. The origin of replication and the chloramphenicol resistance gene on pRF28 were used to construct cloning vectors that are capable of replication in R. fascians and Escherichia coli. The electroporation method presented was efficient enough to allow detection of the rare integration of replication-deficient pRF28 derivatives in the R.fascians D188 genome via either homologous or illegitimate recombination.

  15. Identification of Rhodococcus fascians cytokinins and their modus operandi to reshape the plant.

    PubMed

    Pertry, Ine; Václavíková, Katerina; Depuydt, Stephen; Galuszka, Petr; Spíchal, Lukás; Temmerman, Wim; Stes, Elisabeth; Schmülling, Thomas; Kakimoto, Tatsuo; Van Montagu, Marc C E; Strnad, Miroslav; Holsters, Marcelle; Tarkowski, Petr; Vereecke, Danny

    2009-01-20

    Decades ago, the importance of cytokinins (CKs) during Rhodococcus fascians pathology had been acknowledged, and an isopentenyltransferase gene had been characterized in the fas operon of the linear virulence plasmid, but hitherto, no specific CK(s) could be associated with virulence. We show that the CK receptors AHK3 and AHK4 of Arabidopsis thaliana are essential for symptom development, and that the CK perception machinery is induced upon infection, underlining its central role in the symptomatology. Three classical CKs [isopentenyladenine, trans-zeatin, and cis-zeatin (cZ)] and their 2-methylthio (2MeS)-derivatives were identified by CK profiling of both the pathogenic R. fascians strain D188 and its nonpathogenic derivative D188-5. However, the much higher CK levels in strain D188 suggest that the linear plasmid is responsible for the virulence-associated production. All R. fascians CKs were recognized by AHK3 and AHK4, and, although they individually provoked typical CK responses in several bioassays, the mixture of bacterial CKs exhibited clear synergistic effects. The cis- and 2MeS-derivatives were poor substrates of the apoplastic CK oxidase/dehydrogenase enzymes and the latter were not cytotoxic at high concentrations. Consequently, the accumulating 2MeScZ (and cZ) in infected Arabidopsis tissue contribute to the continuous stimulation of tissue proliferation. Based on these results, we postulate that the R. fascians pathology is based on the local and persistent secretion of an array of CKs.

  16. Phenotypic mutants of the intracellular actinomycete Rhodococcus equi created by in vivo Himar1 transposon mutagenesis.

    PubMed

    Ashour, Joseph; Hondalus, Mary K

    2003-04-01

    Rhodococcus equi is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of R. equi disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a Himar1 minitransposon delivered by a conditionally replicating plasmid for construction of R. equi mutants. We show that Himar1 transposition in R. equi is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of R. equi. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type R. equi, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of R. equi virulence mechanisms and the creation of other attenuated strains with vaccine potential.

  17. Biofilm formation and partial biodegradation of polystyrene by the actinomycete Rhodococcus ruber: biodegradation of polystyrene.

    PubMed

    Mor, Roi; Sivan, Alex

    2008-11-01

    Polystyrene, which is one of the most utilized thermoplastics, is highly durable and is considered to be non-biodegradable. Hence, polystyrene waste accumulates in the environment posing an increasing ecological threat. In a previous study we have isolated a biofilm-producing strain (C208) of the actinomycete Rhodococcus ruber that degraded polyethylene films. Formation of biofilm, by C208, improved the biodegradation of polyethylene. Consequently, the present study aimed at monitoring the kinetics of biofilm formation by C208 on polystyrene, determining the physiological activity of the biofilm and analyzing its capacity to degrade polystyrene. Quantification of the biofilm biomass was performed using a modified crystal violet (CV) staining or by monitoring the protein content in the biofilm. When cultured on polystyrene flakes, most of the bacterial cells adhered to the polystyrene surface within few hours, forming a biofilm. The growth of the on polystyrene showed a pattern similar to that of a planktonic culture. Furthermore, the respiration rate, of the biofilm, exhibited a pattern similar to that of the biofilm growth. In contrast, the respiration activity of the planktonic population showed a constant decline with time. Addition of mineral oil (0.005% w/v), but not non-ionic surfactants, increased the biofilm biomass. Extended incubation of the biofilm for up to 8 weeks resulted in a small reduction in the polystyrene weight (0.8% of gravimetric weight loss). This study demonstrates the high affinity of C208 to polystyrene which lead to biofilm formation and, presumably, induced partial biodegradation.

  18. Trehalose promotes Rhodococcus sp. strain YYL colonization in activated sludge under tetrahydrofuran (THF) stress

    PubMed Central

    He, Zhixing; Zhang, Kai; Wang, Haixia; Lv, Zhenmei

    2015-01-01

    Few studies have focused on the role of compatible solutes in changing the microbial community structure in bioaugmentation systems. In this study, we investigated the influence of trehalose as a biostimulant on the microbial community in tetrahydrofuran (THF)-treated wastewater bioaugmentation systems with Rhodococcus sp. YYL. Functional gene profile changes were used to study the variation in the microbial community. Soluble di-iron monooxygenases (SDIMO), particularly group-5 SDIMOs (i.e., tetrahydrofuran and propane monooxygenases), play a significant role in the initiation of the ring cleavage of tetrahydrofuran. Group-5 SDIMOs genes are enriched upon trehalose addition, and exogenous tetrahydrofuran monooxygenase (thmA) genes can successfully colonize bioaugmentation systems. Cytochrome P450 monooxygenases (P450s) have a significant role in catalyzing the region- and stereospecific oxidation of non-activated hydrocarbons, and THF was reported to inhibit P450s in the environment. The CYP153 family was chosen as a representative P450 to study the inhibitory effects of THF. The results demonstrated that CYP153 family genes exhibited significant changes upon THF treatment and that trehalose helped maintain a rich diversity and high abundance of CYP153 family genes. Biostimulation with trehalose could alleviate the negative effects of THF stress on microbial diversity in bioaugmentation systems. Our results indicated that trehalose as a compatible solute plays a significant role for environmental strains under extreme conditions. PMID:26029182

  19. Response of Rhodococcus erythropolis strain IBBPo1 to toxic organic solvents

    PubMed Central

    Stancu, Mihaela Marilena

    2015-01-01

    Abstract Recently, there has been a lot of interest in the utilization of rhodococci in the bioremediation of petroleum contaminated environments. This study investigates the response of Rhodococcus erythropolis IBBPo1 cells to 1% organic solvents (alkanes, aromatics). A combination of microbiology, biochemical, and molecular approaches were used to examine cell adaptation mechanisms likely to be pursued by this strain after 1% organic solvent exposure. R. erythropolis IBBPo1 was found to utilize 1% alkanes (cyclohexane, n-hexane, n-decane) and aromatics (toluene, styrene, ethylbenzene) as the sole carbon source. Modifications in cell viability, cell morphology, membrane permeability, lipid profile, carotenoid pigments profile and 16S rRNA gene were revealed in R. erythropolis IBBPo1 cells grown 1 and 24 h on minimal medium in the presence of 1% alkanes (cyclohexane, n-hexane, n-decane) and aromatics (toluene, styrene, ethylbenzene). Due to its environmental origin and its metabolic potential, R. erythropolis IBBPo1 is an excellent candidate for the bioremediation of soils contaminated with crude oils and other toxic compounds. Moreover, the carotenoid pigments produced by this nonpathogenic Gram-positive bacterium have a variety of other potential applications. PMID:26691458

  20. A Long-Chain Secondary Alcohol Dehydrogenase from Rhodococcus erythropolis ATCC 4277

    PubMed Central

    Ludwig, B.; Akundi, A.; Kendall, K.

    1995-01-01

    A NAD-dependent secondary alcohol dehydrogenase has been purified from the alkane-degrading bacterium, Rhodococcus erythropolis ATCC 4277. The enzyme was found to be active against a broad range of substrates, particularly long-chain secondary aliphatic alcohols. Although optimal activity was observed with linear 2-alcohols containing between 6 and 11 carbon atoms, secondary alcohols as long as 2-tetradecanol were oxidized at 25% of the rate seen with mid-range alcohols. The purified enzyme was specific for the S-(+) stereoisomer of 2-octanol and had a specific activity for 2-octanol of over 200 (mu)mol/min/mg of protein at pH 9 and 37(deg)C, 25-fold higher than that of any previously reported S-(+) secondary alcohol dehydrogenase. Linear primary alcohols containing between 3 and 13 carbon atoms were utilized 20- to 40-fold less efficiently than the corresponding secondary alcohols. The apparent K(infm) value for NAD(sup+) with 2-octanol as the substrate was 260 (mu)M, whereas the apparent K(infm) values for the 2-alcohols ranged from over 5 mM for 2-pentanol to less than 2 (mu)M for 2-tetradecanol. The enzyme showed moderate thermostability (half-life of 4 h at 60(deg)C) and could potentially be useful for the synthesis of optically pure stereoisomers of secondary alcohols. PMID:16535152

  1. A bifunctional enzyme from Rhodococcus erythropolis exhibiting secondary alcohol dehydrogenase-catalase activities.

    PubMed

    Martinez-Rojas, Enriqueta; Kurt, Tutku; Schmidt, Udo; Meyer, Vera; Garbe, Leif-Alexander

    2014-11-01

    Alcohol dehydrogenases have long been recognized as potential biocatalyst for production of chiral fine and bulk chemicals. They are relevant for industry in enantiospecific production of chiral compounds. In this study, we identified and purified a nicotinamide adenine dinucleotide (NAD)-dependent secondary alcohol dehydrogenase (SdcA) from Rhodococcus erythropolis oxidizing γ-lactols into γ-lactones. SdcA showed broad substrate specificity on γ-lactols; secondary aliphatic alcohols with 8 and 10 carbon atoms were also substrates and oxidized with (2S)-stereospecificity. The enzyme exhibited moderate stability with a half-life of 5 h at 40 °C and 20 days at 4 °C. Mass spectrometric identification revealed high sequence coverage of SdcA amino acid sequence to a highly conserved catalase from R. erythropolis. The corresponding encoding gene was isolated from genomic DNA and subsequently overexpressed in Escherichia coli BL21 DE3 cells. In addition, the recombinant SdcA was purified and characterized in order to confirm that the secondary alcohol dehydrogenase and catalase activity correspond to the same enzyme.

  2. Rhodococcus sp. Q5, a novel agarolytic bacterium isolated from printing and dyeing wastewater.

    PubMed

    Feng, Zehua; Peng, Lin; Chen, Mei; Li, Mengying

    2012-09-01

    An agar-degrading bacterium, Rhodococcus sp. Q5, was isolated from printing and dyeing wastewater using a mineral salts agar plate containing agar as the sole carbon source. The bacterium grew from pH 4.0 to 9.0, from 15 to 35°C, and in NaCl concentrations of 0-5 %; optimal values were pH 6.0, 30°C, and 1 % NaCl. Maximal agarase production was observed at pH 6.0 and 30°C. The bacterium did not require NaCl for growth or agarase production. The agarase secreted by Q5 was inducible by agar and was repressed by all simple sugars tested except lactose. Strain Q5 could hydrolyze starch but not cellulose or carboxymethyl cellulose. Agarase activity could also be detected in the medium when lactose or starch was the sole source of carbon and energy. Strain Q5 could grow in nitrogen-free mineral media; an organic nitrogen source was more effective than inorganic carbon sources for growth and agarase production. Addition of more organic nitrogen (peptone) to the medium corresponded with reduced agarase activity.

  3. Degradation of chloronitrobenzenes by a coculture of Pseudomonas putida and a Rhodococcus sp.

    SciTech Connect

    Park, H.S.; Lim, S.J.; Chang, Y.K.; Kim, H.S.; Livingston, A.G.

    1999-03-01

    A single microorganism able to mineralize chloronitrobenzenes (CNBs) has not been reported, and degradation of CNBs of coculture of two microbial strains was attempted. Pseudomonas putida HS12 was first isolated by analogue enrichment culture using nitrobenzene (NB) as the substrate, and this strain was observed to possess a partial reductive pathway for the degradation of NB. From high-performance liquid chromatography-mass spectrometry and {sup 1}H nuclear magnetic resonance analyses, NB-grown cells of P. putida HS12 were found to convert 3- and 4-CNBs to the corresponding 5- and 4-chloro-2-hydroxyacetanilides, respectively, by partial reduction and subsequent acetylation. For the degradation of CNBs, Rhodococcus sp. strain HS51, which degrades 4- and 5-chloro-2-hydroxyacetanilides, was isolated and combined with P. putida HS12 to give a coculture. This coculture was confirmed to mineralize 3- and 4-CNBs in the presence of an additional carbon source. A degradation pathway for 3- and 4-CNBs by the two isolated strains was also proposed.

  4. Virulence quenching with a prenylated isoflavanone renders the Malagasy legume Dalbergia pervillei resistant to Rhodococcus fascians.

    PubMed

    Rajaonson, Sanda; Vandeputte, Olivier M; Vereecke, Danny; Kiendrebeogo, Martin; Ralambofetra, Eliane; Stévigny, Caroline; Duez, Pierre; Rabemanantsoa, Christian; Mol, Adeline; Diallo, Billo; Baucher, Marie; El Jaziri, Mondher

    2011-05-01

    The phytopathogenic Actinomycete Rhodococcus fascians induces leafy galls on a wide range of hosts, causing major economical losses in the ornamentals industry. Although differences in the responsivity occur within species, no plant tested so far could be considered resistant to R. fascians strain D188 infection. Here, we observed that members of the genus Dalbergia, which belong to the Fabaceae, did not develop leafy galls when challenged with R. fascians and we set out to unravel the mechanism of this recalcitrance. Whereas organic extracts of Dalbergia tissues exhibited toxicity towards the bacteria, more importantly, dichloromethane bark extracts inhibited the induction of bacterial virulence gene expression without any apparent loss of viability, illustrating that resistance is likely multifactorial. The virulence quencher was identified as a new prenylated isoflavanone, termed perbergin, and specifically targeted the AttR regulon (a LysR-type transcriptional regulator) which is imperative for the switch of R. fascians from an epiphytic to a pathogenic lifestyle. The mode of action of perbergin demonstrated that just like in Gram-negative host-microbe interactions, also in Gram-positive phytopathogens autoregulation is being targeted by the plant as an efficient means of defence. Moreover, the identification of perbergin opens the path to disease control in affected nurseries.

  5. Identification of novel extracellular protein for PCB/biphenyl metabolism in Rhodococcus jostii RHA1.

    PubMed

    Atago, Yuki; Shimodaira, Jun; Araki, Naoto; Bin Othman, Nor'azizi; Zakaria, Zuriati; Fukuda, Masao; Futami, Junichiro; Hara, Hirofumi

    2016-05-01

    Rhodococcus jostii RHA1 (RHA1) degrades polychlorinated biphenyl (PCB) via co-metabolism with biphenyl. To identify the novel open reading frames (ORFs) that contribute to PCB/biphenyl metabolism in RHA1, we compared chromatin immunoprecipitation chip and transcriptomic data. Six novel ORFs involved in PCB/biphenyl metabolism were identified. Gene deletion mutants of these 6 ORFs were made and were tested for their ability to grow on biphenyl. Interestingly, only the ro10225 deletion mutant showed deficient growth on biphenyl. Analysis of Ro10225 protein function showed that growth of the ro10225 deletion mutant on biphenyl was recovered when exogenous recombinant Ro10225 protein was added to the culture medium. Although Ro10225 protein has no putative secretion signal sequence, partially degraded Ro10225 protein was detected in conditioned medium from wild-type RHA1 grown on biphenyl. This Ro10225 fragment appeared to form a complex with another PCB/biphenyl oxidation enzyme. These results indicated that Ro10225 protein is essential for the formation of the PCB/biphenyl dioxygenase complex in RHA1.

  6. Deep desulfurization of diesel oil and crude oils by a newly isolated Rhodococcus erythropolis strain.

    PubMed

    Yu, Bo; Xu, Ping; Shi, Quan; Ma, Cuiqing

    2006-01-01

    The soil-isolated strain XP was identified as Rhodococcus erythropolis. R. erythropolis XP could efficiently desulfurize benzonaphthothiophene, a complicated model sulfur compound that exists in crude oil. The desulfurization product of benzonaphthothiophene was identified as alpha-hydroxy-beta-phenyl-naphthalene. Resting cells could desulfurize diesel oil (total organic sulfur, 259 ppm) after hydrodesulfurization. The sulfur content of diesel oil was reduced by 94.5% by using the resting cell biocatalyst for 24 h at 30 degrees C. Biodesulfurization of crude oils was also investigated. After 72 h of treatment at 30 degrees C, 62.3% of the total sulfur content in Fushun crude oil (initial total sulfur content, 3,210 ppm) and 47.2% of that in Sudanese crude oil (initial total sulfur, 1,237 ppm) were removed. Gas chromatography with pulsed-flame photometric detector analysis was used to evaluate the effect of R. erythropolis XP treatment on the sulfur content in Fushun crude oil, and it was shown that most organic sulfur compounds were eliminated after biodesulfurization.

  7. Immunogenicity of an Electron Beam Inactivated Rhodococcus equi Vaccine in Neonatal Foals

    PubMed Central

    Bordin, Angela I.; Pillai, Suresh D.; Brake, Courtney; Bagley, Kaytee B.; Bourquin, Jessica R.; Coleman, Michelle; Oliveira, Fabiano N.; Mwangi, Waithaka; McMurray, David N.; Love, Charles C.; Felippe, Maria Julia B.; Cohen, Noah D.

    2014-01-01

    Rhodococcus equi is an important pathogen of foals that causes severe pneumonia. To date, there is no licensed vaccine effective against R. equi pneumonia of foals. The objectives of our study were to develop an electron beam (eBeam) inactivated vaccine against R. equi and evaluate its immunogenicity. A dose of eBeam irradiation that inactivated replication of R. equi while maintaining outer cell wall integrity was identified. Enteral administration of eBeam inactivated R. equi increased interferon-γ production by peripheral blood mononuclear cells in response to stimulation with virulent R. equi and generated naso-pharyngeal R. equi-specific IgA in newborn foals. Our results indicate that eBeam irradiated R. equi administered enterally produce cell-mediated and upper respiratory mucosal immune responses, in the face of passively transferred maternal antibodies, similar to those produced in response to enteral administration of live organisms (a strategy which previously has been documented to protect foals against intrabronchial infection with virulent R. equi). No evidence of adverse effects was noted among vaccinated foals. PMID:25153708

  8. Perchlorate reduction from a highly concentrated aqueous solution by bacterium Rhodococcus sp. YSPW03.

    PubMed

    Lee, Sang-Hoon; Hwang, Jae-Hoon; Kabra, Akhil N; Abou-Shanab, Reda A I; Kurade, Mayur B; Min, Booki; Jeon, Byong-Hun

    2015-12-01

    A novel isolated bacterium Rhodococcus sp. YSPW03 was able to reduce high concentrations (up to 700 mg L(-1)) of perchlorate using acetate as electron donor. Perchlorate reduction rate increased from 2.90 to 11.23 mg L(-1) h(-1) with increasing initial acetate concentration from 100 to 2000 mg L(-1), leading to complete removal of perchlorate (100 mg L(-1)) within 9 h. The bacterium also promoted complete reduction of high perchlorate concentrations (500 and 700 mg L(-1)) at 2000 mg L(-1) of acetate within 48 and 96 h, respectively. Under semi-continuous reactor operation, efficient reduction on varied perchlorate concentrations (80-700 mg L(-1)) was performed by the bacterium in presence of acetate (600-6000 mg L(-1)) over 140 days. The highest perchlorate reduction rate of 280 mg L(-1) day(-1) was observed with an initial perchlorate concentration of 570 mg L(-1) at day 34. Dissolved chloride ions of 1000 mg L(-1) in the semi-continuous reactor (SCR) completely inhibited the biological perchlorate reduction. The findings of this study will help improve the perchlorate bioreactor design and determine the optimal conditions to maximize the perchlorate reduction efficiency.

  9. Prevention of foal mortality due to Rhodococcus equi pneumonia on an endemically affected farm

    PubMed Central

    Prescott, John F.; Machang'u, Robert; Kwiecien, Jacek; Delaney, Kathy

    1989-01-01

    Over the course of one summer, foals on a horse breeding farm where Rhodococcus equi infection was endemic were examined clinically twice weekly for evidence of R. equi pneumonia. Examination usually commenced from the week of birth and continued for up to 14 weeks of age. Affected animals were treated with a variety of antimicrobial drugs and such treatment was often prolonged. For descriptive purposes, we regarded a foal as developing R. equi pneumonia if the rectal temperature rose above 39°C, the respiratory rate was over 40 per min, there were characteristic associated changes in respiratory sounds on auscultation, and the foal responded to antimicrobial treatment. In a group of 16 foals followed in this way, R. equi pneumonia developed in at least 14. Seven of these foals developed antibodies to equi factors as determined in a neutralizing antibody assay. Mean age of onset of these 14 foals was 3.4 weeks. Three foals developed the disease between 1.5-2.5 weeks of age, and 12 of 14 before four weeks of age. In previous years four to six foals died on the farm each summer of R. equi pneumonia; only one foal died of R. equi pneumonia on the farm during the summer of this study, and this foal did not form part of the study group. Early clinical recognition and treatment of R. equi pneumonia in foals on endemically affected farms may be an effective way to prevent deaths due to this infection. PMID:17423454

  10. Lignocellulose-derived inhibitors improve lipid extraction from wet Rhodococcus opacus cells.

    PubMed

    Kurosawa, Kazuhiko; Anthony Debono, C; Sinskey, Anthony J

    2015-10-01

    Extracting lipids from oleaginous microbial cells in a cost effective and environmentally compatible manner remains a critical challenge in developing manufacturing paradigms for advanced liquid biofuels. In this study, a new approach using microbial growth inhibitors from lignocellulose-derived feedstocks was used to extract lipids efficiently from wet cell mass of the oleaginous bacterium Rhodococcus opacus MITXM-61. Nine common lignocellulose-derived inhibitors for treatment of cells prior to solvent extraction were used and evaluated for their efficiency of lipid extraction from the cells. When the inhibitors were individually examined, formic acid and furfural showed the highest extraction efficiency of lipids from wet cell mass. Multiple extractions of lipids with methanol from wet cell mass pretreated with combined common inhibitors or hardwood hydrolysate comprising lignocellulose-derived inhibitors resulted in lipid recovery of greater than 85% of total lipids, a 1.7-fold increase of lipid extraction as compared to those in the absence of the inhibitors. Copyright © 2015. Published by Elsevier Ltd.

  11. Engineering L-arabinose metabolism in triacylglycerol-producing Rhodococcus opacus for lignocellulosic fuel production.

    PubMed

    Kurosawa, Kazuhiko; Plassmeier, Jens; Kalinowski, Jörn; Rückert, Christian; Sinskey, Anthony J

    2015-07-01

    Advanced biofuels from lignocellulosic biomass have been considered as a potential solution for the issues of energy sustainability and environmental protection. Triacylglycerols (TAGs) are potential precursors for the production of lipid-based liquid biofuels. Rhodococcus opacus PD630 can accumulate large amounts of TAGs when grown under physiological conditions of high carbon and low nitrogen. However, R. opacus PD630 does not utilize the sugar L-arabinose present in lignocellulosic hydrolysates. Here, we report the engineering of R. opacus to produce TAGs on L-arabinose. We constructed a plasmid (pASC8057) harboring araB, araD and araA genes derived from a Streptomyces bacterium, and introduced the genes into R. opacus PD630. One of the engineered strains, MITAE-348, was capable of growing on high concentrations (up to 100 g/L) of L-arabinose. MITAE-348 was grown in a defined medium containing 16 g/L L-arabinose or a mixture of 8 g/L L-arabinose and 8 g/L D-glucose. In a stationary phase occurring 3 days post-inoculation, the strain was able to completely utilize the sugar, and yielded 2.0 g/L for L-arabinose and 2.2 g/L for L-arabinose/D-glucose of TAGs, corresponding to 39.7% or 42.0%, respectively, of the cell dry weight. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  12. Comparative transcriptomics elucidates adaptive phenol tolerance and utilization in lipid-accumulating Rhodococcus opacus PD630

    PubMed Central

    Yoneda, Aki; Henson, William R.; Goldner, Nicholas K.; Park, Kun Joo; Forsberg, Kevin J.; Kim, Soo Ji; Pesesky, Mitchell W.; Foston, Marcus; Dantas, Gautam; Moon, Tae Seok

    2016-01-01

    Lignin-derived (e.g. phenolic) compounds can compromise the bioconversion of lignocellulosic biomass to fuels and chemicals due to their toxicity and recalcitrance. The lipid-accumulating bacterium Rhodococcus opacus PD630 has recently emerged as a promising microbial host for lignocellulose conversion to value-added products due to its natural ability to tolerate and utilize phenolics. To gain a better understanding of its phenolic tolerance and utilization mechanisms, we adaptively evolved R. opacus over 40 passages using phenol as its sole carbon source (up to 373% growth improvement over wild-type), and extensively characterized two strains from passages 33 and 40. The two adapted strains showed higher phenol consumption rates (∼20 mg/l/h) and ∼2-fold higher lipid production from phenol than the wild-type strain. Whole-genome sequencing and comparative transcriptomics identified highly-upregulated degradation pathways and putative transporters for phenol in both adapted strains, highlighting the important linkage between mechanisms of regulated phenol uptake, utilization, and evolved tolerance. Our study shows that the R. opacus mutants are likely to use their transporters to import phenol rather than export them, suggesting a new aromatic tolerance mechanism. The identified tolerance genes and pathways are promising candidates for future metabolic engineering in R. opacus for improved lignin conversion to lipid-based products. PMID:26837573

  13. 2D-crystallization of Rhodococcus 20S proteasome at the liquid-liquid interface

    NASA Astrophysics Data System (ADS)

    Aoyama, Kazuhiro

    1996-10-01

    The 2D-crystallization method using the liquid-liquid interface between a aqueous phase (protein solution) and a thin organic liquid (dehydroabietylamine) layer has been applied to the Rhodococcus 20S proteasome. The 20S proteasome is known to be the core complex of the 26S proteasome, which is the central protease of the ubiquitin-dependent pathway. Two types of ordered arrays were obtained, both large enough for high resolution analysis by electron crystallography. The first one had a four-fold symmetry, whereas the second one was found out to be a hexagonally close-packed array. By image analysis based on a real space correlation averaging (CAV) technique, the close-packed array was found to be hexagonally packed, but the molecules had presumably rotational freedom. The four-fold array was found to be a true crystal with p4 symmetry. Lattice constants were a = b = 20.0 nm and α = 90°. The unit cell of this crystal contained two molecules. The diffraction pattern computed from the original picture showed spots up to (4, 5) that corresponds to 3.1 nm resolution. After applying an unbending procedure, the diffraction pattern showed spots extending to 1.8 nm resolution.

  14. Identification and characterization of genes involved in naphthalene degradation in Rhodococcus opacus R7.

    PubMed

    Di Gennaro, Patrizia; Terreni, Paola; Masi, Gianmarco; Botti, Silvia; De Ferra, Francesca; Bestetti, Giuseppina

    2010-06-01

    Rhodococcus opacus R7 is a naphthalene-degrading microorganism which is also able to grow on o-xylene. This work describes the isolation and analysis of two new genomic regions in which genes involved in naphthalene (nar gene cluster) and salicylate (gen gene cluster) degradation are located. In the nar gene cluster we found: two genes encoding the large (narAa) and the small (narAb) components of the naphthalene dioxygenase, three genes (rub1, rub2, rub1bis) encoding three rubredoxins, an orf (orf7) associated to the complex encoding a protein of unknown function, two regulatory genes (narR1, narR2), a gene (narB) encoding the naphthalene dihydrodiol dehydrogenase and six orfs (orf1, orf2, orf3, orf4, orf5, orf6) encoding proteins of unknown function. In the gen gene cluster, we found the following genes: two genes encoding the salicylate CoA ligase and the salicylate CoA synthetase (genA and genB), respectively, a gene (genC) encoding a salicylate hydroxylase, a gene (genH) encoding a gentisate 1,2-dioxygenase, a gene (genI) encoding a 3-maleylpyruvate isomerase, and a gene (genL) encoding a protein of unknown function. The transcription of some genes of R. opacus R7 strain grown on different substrates was also investigated to evaluate the expression of the two gene clusters after cDNA preparations.

  15. Biotransformation of 1,3-propanediol cyclic sulfate and its derivatives to diols by Rhodococcus sp.

    PubMed

    He, Yu-Cai; Tao, Zhi-Cheng; Zhang, Dan-Ping; Yang, Zhen-Xing; Gao, Shan; Ma, Cui-Luan

    2015-01-01

    Rhodococcus sp. CGMCC 4911 transformed 1,3-propanediol cyclic sulfate (1,3-PDS) and its derivatives into corresponding diols. Ethylene sulfate, glycol sulfide, 1,3-PDS, and 1,2-propanediol cyclic sulfate were effectively hydrolyzed with growing cells. (R)-1,2-Propanediol (>99 % e.e.) was obtained at 44 % yield with growing cells. Glycol sulfide, ethylene sulfate, and 1,3-PDS were converted into the corresponding diols at 94.6, 96.3, and 98.3 %, respectively. Optimal reaction conditions with lyophilized resting cells were 30 °C, pH 7.5, and cell dosage 17.9 mg cell dry wt/ml. 1,3-Propanediol was obtained from 50 mM 1,3-PDS at 97.2 % yield by lyophilized cells after 16 h. Lyophilized cells were entrapped in calcium alginate with a half-life of 263 h at 30 °C, and the total operational time of the immobilized biocatalysts could reach over 192 h with a high conversion rate.

  16. Discovery and characterization of a putrescine oxidase from Rhodococcus erythropolis NCIMB 11540

    PubMed Central

    van Hellemond, Erik W.; van Dijk, Marianne; Heuts, Dominic P. H. M.; Janssen, Dick B.

    2008-01-01

    A gene encoding a putrescine oxidase (PuORh, EC 1.4.3.10) was identified from the genome of Rhodococcus erythropolis NCIMB 11540. The gene was cloned in the pBAD vector and overexpressed at high levels in Escherichia coli. The purified enzyme was shown to be a soluble dimeric flavoprotein consisting of subunits of 50 kDa and contains non-covalently bound flavin adenine dinucleotide as a cofactor. From all substrates, the highest catalytic efficiency was found with putrescine (KM = 8.2 μM, kcat = 26 s−1). PuORh accepts longer polyamines, while short diamines and monoamines strongly inhibit activity. PuORh is a reasonably thermostable enzyme with t1/2 at 50°C of 2 h. Based on the crystal structure of human monoamine oxidase B, we constructed a model structure of PuORh, which hinted to a crucial role of Glu324 for substrate binding. Mutation of this residue resulted in a drastic drop (five orders of magnitude) in catalytic efficiency. Interestingly, the mutant enzyme showed activity with monoamines, which are not accepted by wt-PuORh. Electronic supplementary material The online version of this article (doi:10.1007/s00253-007-1310-4) contains supplementary material, which is available to authorized users. PMID:18183391

  17. Engineering levoglucosan metabolic pathway in Rhodococcus jostii RHA1 for lipid production.

    PubMed

    Xiong, Xiaochao; Lian, Jieni; Yu, Xiaochen; Garcia-Perez, Manuel; Chen, Shulin

    2016-11-01

    Oleaginous strains of Rhodococcus including R. jostii RHA1 have attracted considerable attention due to their ability to accumulate triacylglycerols (TAGs), robust growth properties and genetic tractability. In this study, a novel metabolic pathway was introduced into R. jostii by heterogenous expression of the well-characterized gene, lgk encoding levoglucosan kinase from Lipomyces starkeyi YZ-215. This enables the recombinant R. jostii RHA1 to produce TAGs from the anhydrous sugar, levoglucosan, which can be generated efficiently as the major molecule from the pyrolysis of cellulose. The recombinant R. jostii RHA1 could grow on levoglucosan as the sole carbon source, and the consumption rate of levoglucosan was determined. Furthermore, expression of one more copy of lgk increased the enzymatic activity of LGK in the recombinant. However, the growth performance of the recombinant bearing two copies of lgk on levoglucosan was not improved. Although expression of lgk in the recombinants was not repressed by the glucose present in the media, glucose in the sugar mixture still affected consumption of levoglucosan. Under nitrogen limiting conditions, lipid produced from levoglucosan by the recombinant bearing lgk was up to 43.54 % of the cell dry weight, which was comparable to the content of lipid accumulated from glucose. This work demonstrated the technical feasibility of producing lipid from levoglucosan, an anhydrosugar derived from the pyrolysis of lignocellulosic materials, by the genetically modified rhodococci strains.

  18. Rhodococcus equi infection in HIV-positive subjects: a retrospective analysis of 24 cases.

    PubMed

    Arlotti, M; Zoboli, G; Moscatelli, G L; Magnani, G; Maserati, R; Borghi, V; Andreoni, M; Libanore, M; Bonazzi, L; Piscina, A; Ciammarughi, R

    1996-01-01

    Rhodococcus equi causes a rare infection in immunocompromised hosts. We describe 24 cases of infection in patients with AIDS-related complex (ARC)/acquired immunodeficiency syndrome (AIDS). Pneumonia was always the first manifestation of R. equi infection, but extrapulmonary involvement was also observed. The main sources of bacteria were sputum, bronchial washings and blood. The strains isolated were mainly susceptible to erythromycin, vancomycin, teicoplanin, rifampicin, imipenem and aminoglycosides. Initial treatment should involve an intravenously administered antibiotic combination therapy including imipenem or vancomycin or teicoplanin, followed by orally administered maintenance combination therapy. Drug combinations should be investigated for serum bactericidal activity in vitro. Surgery does not increase survival time and should only be performed in cases that do not respond to antibiotic treatment. Presumptive risks of infection (contact with horses or farm dust, or cohabiting with people affected by R. equi infection) were present in more than 50% of patients. This finding, and the frequency of bacteria in the sputum, are not sufficient proof of transmission between humans, but do suggest the need for respiratory isolation of patients affected by R. equi pneumonia.

  19. Aerobic Biodegradation of N-Nitrosodimethylamine by the Propanotroph Rhodococcus ruber ENV425▿

    PubMed Central

    Fournier, Diane; Hawari, Jalal; Halasz, Annamaria; Streger, Sheryl H.; McClay, Kevin R.; Masuda, Hisako; Hatzinger, Paul B.

    2009-01-01

    The propanotroph Rhodococcus ruber ENV425 was observed to rapidly biodegrade N-nitrosodimethylamine (NDMA) after growth on propane, tryptic soy broth, or glucose. The key degradation intermediates were methylamine, nitric oxide, nitrite, nitrate, and formate. Small quantities of formaldehyde and dimethylamine were also detected. A denitrosation reaction, initiated by hydrogen atom abstraction from one of the two methyl groups, is hypothesized to result in the formation of n-methylformaldimine and nitric oxide, the former of which decomposes in water to methylamine and formaldehyde and the latter of which is then oxidized further to nitrite and then nitrate. Although the strain mineralized more than 60% of the carbon in [14C]NDMA to 14CO2, growth of strain ENV425 on NDMA as a sole carbon and energy source could not be confirmed. The bacterium was capable of utilizing NDMA, as well as the degradation intermediates methylamine and nitrate, as sources of nitrogen during growth on propane. In addition, ENV425 reduced environmentally relevant microgram/liter concentrations of NDMA to <2 ng/liter in batch cultures, suggesting that the bacterium may have applications for groundwater remediation. PMID:19542346

  20. Phenotypic Mutants of the Intracellular Actinomycete Rhodococcus equi Created by In Vivo Himar1 Transposon Mutagenesis

    PubMed Central

    Ashour, Joseph; Hondalus, Mary K.

    2003-01-01

    Rhodococcus equi is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of R. equi disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a Himar1 minitransposon delivered by a conditionally replicating plasmid for construction of R. equi mutants. We show that Himar1 transposition in R. equi is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of R. equi. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type R. equi, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of R. equi virulence mechanisms and the creation of other attenuated strains with vaccine potential. PMID:12670990

  1. Gamma-caprolactone stimulates growth of quorum-quenching Rhodococcus populations in a large-scale hydroponic system for culturing Solanum tuberosum.

    PubMed

    Cirou, Amélie; Raffoux, Aurélie; Diallo, Stéphanie; Latour, Xavier; Dessaux, Yves; Faure, Denis

    2011-11-01

    Bacteria degrading quorum sensing (QS) signals have been proposed as biocontrol agents able to quench QS-dependent expression of virulence symptoms caused by Pectobacterium on potato plants. We report here that gamma-caprolactone (GCL) treatment stimulated growth of the native QS-degrading bacterial community in an industrial plant hydroponic system for culturing Solanum tuberosum. Post-GCL treatment, QS-degrading bacteria were mainly identified as Rhodococcus isolates, while Agrobacterium isolates dominated under similar untreated conditions. Most of the assayed Rhodococcus isolates exhibited efficient biocontrol activity for protecting potato tubers. Analytical chemistry approach revealed the rapid degradation of GCL introduced in the plant cultures.

  2. Functional divergence of HBHA from Mycobacterium tuberculosis and its evolutionary relationship with TadA from Rhodococcus opacus.

    PubMed

    Lanfranconi, Mariana P; Alvarez, Héctor M

    2016-08-01

    Rhodococcus opacus PD630 and Rhodococcus jostii RHA1 are oleaginous bacteria able to synthesize and accumulate triacylglycerols (TAG) in lipid bodies (LB). Highly relevant to the structure of LB is a protein homologous to heparin-binding hemagglutinin (HBHA) (called TadA in rhodococci), which is a virulence factor found in Mycobacterium tuberculosis. HBHA is an adhesin involved in binding to non-phagocytic cells and extrapulmonary dissemination. We observed a conserved synteny of three genes encoding a transcriptional regulator (TR), the HBHA protein and a membrane protein (MP) between TAG-accumulating actinobacteria belonging to Rhodococcus, Mycobacterium, Nocardia and Dietzia genera, among others. A 354 bp-intergenic spacing containing a SigF-binding site was found between hbha and the TR genes in M. tuberculosis, which was absent in genomes of other investigated actinobacteria. Analyses of available "omic" information revealed that TadA and TR were co-induced in rhodococci under TAG-accumulating conditions; whereas in M. tuberculosis and Mycobacterium smegmatis, HBHA and TR were regulated independently under stress conditions occurring during infection. We also found differences in protein lengths, domain content and distribution between HBHA and TadA proteins from mycobacteria and rhodococci, which may explain their different roles in cells. Based on the combination of results obtained in model actinobacteria, we hypothesize that HBHA and TadA proteins originated from a common ancestor, but later suffered a process of functional divergence during evolution. Thus, rhodococcal TadA probably has maintained its original role; whereas HBHA may have evolved as a virulence factor in pathogenic mycobacteria.

  3. Identification and Cloning of Genes Involved in Specific Desulfurization of Dibenzothiophene by Rhodococcus sp. Strain IGTS8.

    PubMed

    Denome, S A; Olson, E S; Young, K D

    1993-09-01

    The gram-positive bacterium Rhodococcus sp. strain IGTS8 is able to remove sulfur from certain aromatic compounds without breaking carbon-carbon bonds. In particular, sulfur is removed from dibenzothiophene (DBT) to give the final product, 2-hydroxybiphenyl. A genomic library of IGTS8 was constructed in the cosmid vector pLAFR5, but no desulfurization phenotype was imparted to Escherichia coli. Therefore, IGTS8 was mutagenized, and a new strain (UV1) was selected that had lost the ability to desulfurize DBT. The genomic library was transferred into UV1, and several colonies that had regained the desulfurization phenotype were isolated, though free plasmid could not be isolated. Instead, vector DNA had integrated into either the chromosome or a large resident plasmid. DNA on either side of the inserted vector sequences was cloned and used to probe the original genomic library in E. coli. This procedure identified individual cosmid clones that, when electroporated into strain UV1, restored desulfurization. When the origin of replication from a Rhodococcus plasmid was inserted, the efficiency with which these clones transformed UV1 increased 20- to 50-fold and they could be retrieved as free plasmids. Restriction mapping and subcloning indicated that the desulfurization genes reside on a 4.0-kb DNA fragment. Finally, the phenotype was transferred to Rhodococcus fascians D188-5, a species normally incapable of desulfurizing DBT. The mutant strain, UV1, and R. fascians produced 2-hydroxybiphenyl from DBT when they contained appropriate clones, indicating that the genes for the entire pathway have been isolated.

  4. Remodulation of central carbon metabolic pathway in response to arsenite exposure in Rhodococcus sp. strain NAU‐1

    PubMed Central

    Jain, Raina; Adhikary, Hemanta; Jha, Sanjay; Jha, Anamika; Kumar, G. Naresh

    2012-01-01

    Summary Arsenite‐tolerant bacteria were isolated from an organic farm of Navsari Agricultural University (NAU), Gujarat, India (Latitude: 20°55′39.04″N; Longitude: 72°54′6.34″E). One of the isolates, NAU‐1 (aerobic, Gram‐positive, non‐motile, coccobacilli), was hyper‐tolerant to arsenite (AsIII, 23 mM) and arsenate (AsV, 180 mM). 16S rRNA gene of NAU‐1 was 99% similar to the 16S rRNA genes of Rhodococcus (Accession No. HQ659188). Assays confirmed the presence of membrane bound arsenite oxidase and cytoplasmic arsenate reductase in NAU‐1. Genes for arsenite transporters (arsB and ACR3(1)) and arsenite oxidase gene (aoxB) were confirmed by PCR. Arsenite oxidation and arsenite efflux genes help the bacteria to tolerate arsenite. Specific activities of antioxidant enzymes (catalase, ascorbate peroxidase, superoxide dismutase and glutathione S‐transferase) increased in dose‐dependent manner with arsenite, whereas glutathione reductase activity decreased with increase in AsIII concentration. Metabolic studies revealed that Rhodococcus NAU‐1 produces excess of gluconic and succinic acids, and also activities of glucose dehydrogenase, phosphoenol pyruvate carboxylase and isocitrate lyase were increased, to cope with the inhibited activities of glucose‐6‐phosphate dehydrogenase, pyruvate dehydrogenase and α‐ketoglutarate dehydrogenase enzymes respectively, in the presence of AsIII. Enzyme assays revealed the increase in direct oxidative and glyoxylate pathway in Rhodococcus NAU‐1 in the presence of AsIII. PMID:23062201

  5. Nitrilase-catalyzed production of pyrazinoic acid, an antimycobacterial agent, from cyanopyrazine by resting cells of Rhodococcus rhodochrous J1.

    PubMed

    Kobayashi, M; Yanaka, N; Nagasawa, T; Yamada, H

    1990-10-01

    Using resting cells of Rhodococcus rhodochrous J1, in which a large amount of nitrilase is induced, a simple and efficient bioconversion process for the production of pyrazinoic acid, an antimycobacterial agent, through catalysis by a nitrilase was developed. The reaction conditions for production of pyrazinoic acid were optimized. Under optimum conditions, 3.5 M cyanopyrazine was converted to pyrazinoic acid, with a molar conversion yield of 100%. The highest yield achieved corresponded to 434 g of pyrazinoic acid per liter of reaction mixture. The synthesized pyrazinoic acid was isolated and identified physico-chemically.

  6. Comparative Genomics and Metabolic Analysis Reveals Peculiar Characteristics of Rhodococcus opacus Strain M213 Particularly for Naphthalene Degradation

    PubMed Central

    Blom, Jochen; Indest, Karl J.; Jung, Carina M.; Stothard, Paul; Bera, Gopal; Green, Stefan J.; Ogram, Andrew

    2016-01-01

    The genome of Rhodococcus opacus strain M213, isolated from a fuel-oil contaminated soil, was sequenced and annotated which revealed a genome size of 9,194,165 bp encoding 8680 putative genes and a G+C content of 66.72%. Among the protein coding genes, 71.77% were annotated as clusters of orthologous groups of proteins (COGs); 55% of the COGs were present as paralog clusters. Pulsed field gel electrophoresis (PFGE) analysis of M213 revealed the presence of three different sized replicons- a circular chromosome and two megaplasmids (pNUO1 and pNUO2) estimated to be of 750Kb 350Kb in size, respectively. Conversely, using an alternative approach of optical mapping, the plasmid replicons appeared as a circular ~1.2 Mb megaplasmid and a linear, ~0.7 Mb megaplasmid. Genome-wide comparative analysis of M213 with a cohort of sequenced Rhodococcus species revealed low syntenic affiliation with other R. opacus species including strains B4 and PD630. Conversely, a closer affiliation of M213, at the functional (COG) level, was observed with the catabolically versatile R. jostii strain RHA1 and other Rhodococcii such as R. wratislaviensis strain IFP 2016, R. imtechensis strain RKJ300, Rhodococcus sp. strain JVH1, and Rhodococcus sp. strain DK17, respectively. An in-depth, genome-wide comparison between these functional relatives revealed 971 unique genes in M213 representing 11% of its total genome; many associating with catabolic functions. Of major interest was the identification of as many as 154 genomic islands (GEIs), many with duplicated catabolic genes, in particular for PAHs; a trait that was confirmed by PCR-based identification of naphthalene dioxygenase (NDO) as a representative gene, across PFGE-resolved replicons of strain M213. Interestingly, several plasmid/GEI-encoded genes, that likely participate in degrading naphthalene (NAP) via a peculiar pathway, were also identified in strain M213 using a combination of bioinformatics, metabolic analysis and gene

  7. Fly-attracting volatiles produced by Rhodococcus fascians and Mycobacterium aurum isolated from myiatic lesions of sheep.

    PubMed

    Khoga, Jamal M; Tóth, Erika; Márialigeti, Károly; Borossay, József

    2002-02-01

    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.

  8. Complete genome sequence of Rhodococcus erythropolis BG43 (DSM 46869), a degrader of Pseudomonas aeruginosa quorum sensing signal molecules.

    PubMed

    Rückert, Christian; Birmes, Franziska S; Müller, Christine; Niewerth, Heiko; Winkler, Anika; Fetzner, Susanne; Kalinowski, Jörn

    2015-10-10

    Rhodococcus erythropolis BG43 was isolated from soil and characterized as a degrader of the quorum sensing signal molecules 2-heptyl-3-hydroxy-4(1H)-quinolone (the Pseudomonas quinolone signal, PQS) and 2-heptyl-4(1H)-quinolone, produced by Pseudomonas aeruginosa. The complete genome of R. erythropolis BG43 consists of a circular chromosome and three plasmids, one of them circular and two linear ones. In total, 6158 protein-coding regions were identified. With this genome sequence, the genetic basis of its quorum-quenching ability and possible biotechnological applications can be explored further.

  9. Improved-high-quality draft genome sequence of Rhodococcus sp. JG-3, a eurypsychrophilic Actinobacteria from Antarctic Dry Valley permafrost.

    PubMed

    Goordial, Jacqueline; Raymond-Bouchard, Isabelle; Ronholm, Jennifer; Shapiro, Nicole; Woyke, Tanja; Whyte, Lyle; Bakermans, Corien

    2015-01-01

    The actinobacterium Rhodococcus sp. JG-3 is an aerobic, eurypsychrophilic, soil bacterium isolated from permafrost in the hyper arid Upper Dry Valleys of Antarctica. It is yellow pigmented, gram positive, moderately halotolerant and capable of growth from 30 °C down to at least -5 °C. The 5.28 Mb high-quality-draft genome is arranged into 6 scaffolds, containing 9 contigs and 4998 protein coding genes, with 64 % GC content. Increasing the availability of genome sequences from cold-adapted species is crucial to gaining a better understanding of the molecular traits of cold adaptation in microbes.

  10. Comparative Genomics and Metabolic Analysis Reveals Peculiar Characteristics of Rhodococcus opacus Strain M213 Particularly for Naphthalene Degradation.

    PubMed

    Pathak, Ashish; Chauhan, Ashvini; Blom, Jochen; Indest, Karl J; Jung, Carina M; Stothard, Paul; Bera, Gopal; Green, Stefan J; Ogram, Andrew

    2016-01-01

    The genome of Rhodococcus opacus strain M213, isolated from a fuel-oil contaminated soil, was sequenced and annotated which revealed a genome size of 9,194,165 bp encoding 8680 putative genes and a G+C content of 66.72%. Among the protein coding genes, 71.77% were annotated as clusters of orthologous groups of proteins (COGs); 55% of the COGs were present as paralog clusters. Pulsed field gel electrophoresis (PFGE) analysis of M213 revealed the presence of three different sized replicons- a circular chromosome and two megaplasmids (pNUO1 and pNUO2) estimated to be of 750Kb 350Kb in size, respectively. Conversely, using an alternative approach of optical mapping, the plasmid replicons appeared as a circular ~1.2 Mb megaplasmid and a linear, ~0.7 Mb megaplasmid. Genome-wide comparative analysis of M213 with a cohort of sequenced Rhodococcus species revealed low syntenic affiliation with other R. opacus species including strains B4 and PD630. Conversely, a closer affiliation of M213, at the functional (COG) level, was observed with the catabolically versatile R. jostii strain RHA1 and other Rhodococcii such as R. wratislaviensis strain IFP 2016, R. imtechensis strain RKJ300, Rhodococcus sp. strain JVH1, and Rhodococcus sp. strain DK17, respectively. An in-depth, genome-wide comparison between these functional relatives revealed 971 unique genes in M213 representing 11% of its total genome; many associating with catabolic functions. Of major interest was the identification of as many as 154 genomic islands (GEIs), many with duplicated catabolic genes, in particular for PAHs; a trait that was confirmed by PCR-based identification of naphthalene dioxygenase (NDO) as a representative gene, across PFGE-resolved replicons of strain M213. Interestingly, several plasmid/GEI-encoded genes, that likely participate in degrading naphthalene (NAP) via a peculiar pathway, were also identified in strain M213 using a combination of bioinformatics, metabolic analysis and gene

  11. Microbial Desulfurization of Alkylated Dibenzothiophenes from a Hydrodesulfurized Middle Distillate by Rhodococcus erythropolis I-19

    PubMed Central

    Folsom, B. R.; Schieche, D. R.; DiGrazia, P. M.; Werner, J.; Palmer, S.

    1999-01-01

    Rhodococcus erythropolis I-19, containing multiple copies of key dsz genes, was used to desulfurize alkylated dibenzothiophenes (Cx-DBTs) found in a hydrodesulfurized middle-distillate petroleum (MD 1850). Initial desulfurization rates of dibenzothiophene (DBT) and MD 1850 by I-19 were 5.0 and 2.5 μmol g dry cell weight−1 min−1, more than 25-fold higher than that for wild-type bacteria. According to sulfur K-edge X-ray absorption near-edge structure (XANES) analysis, thiophenic compounds accounted for >95% of the total sulfur found in MD 1850, predominantly Cx-DBTs and alkylated benzothiophenes. Extensive biodesulfurization resulted in a 67% reduction of total sulfur from 1,850 to 615 ppm S. XANES analysis of the 615-ppm material gave a sulfur distribution of 75% thiophenes, 11% sulfides, 2% sulfoxides, and 12% sulfones. I-19 preferentially desulfurized DBT and C1-DBTs, followed by the more highly alkylated Cx-DBTs. Shifting zero- to first-order (first-order) desulfurization rate kinetics were observed when MD 1850 was diluted with hexadecane. Apparent saturation rate constant (K0) and half-saturation rate constant (K1) values were calculated to be 2.8 μmol g dry cell weight−1 min−1 and 130 ppm, respectively. However, partial biocatalytic reduction of MD 1850 sulfur concentration followed by determination of initial rates with fresh biocatalyst led to a sigmoidal kinetic behavior. A competitive-substrate model suggested that the apparent K1 values for each group of Cx-DBTs increased with increasing alkylation. Overall desulfurization rate kinetics with I-19 were affected by the concentration and distribution of Cx-DBTs according to the number and/or lengths of alkyl groups attached to the basic ring structure. PMID:10543810

  12. Seroepidemiological survey of Rhodococcus equi infection in asymptomatic horses and donkeys from southeast Turkey.

    PubMed

    Tel, O Y; Arserim, N B; Keskin, O

    2011-12-01

    In order to assess the level of Rhodococcus equi infection in southeast Turkey, 679 sera from healthy foals and adult horses and 78 sera from donkeys were tested by indirect ELISA using a R. equi reference strain (ATCC 33701) as antigen. Eighty (11.7%) sera from horses and 9 (11.5%) sera from donkeys with titres >0.85 were positive. The prevalence of seropositive horses in Sanliurfa Province was higher than in Diyarbakir Province; 56 (13.9%) horses in Sanliurfa Province and 24 (8.7%) horses in Diyarbakir Province were defined as seropositive. In Sanliurfa Province 14.5% of female (n=343) and 10.1% of male (n = 59) horses tested were defined as seropositive, while in Diyarbakir Province more males (11.4%, n=114) were seropositive than females (6.7%, n=163). Horses 1 to 5 years of age were found to have the highest seropositivity rate in both provinces. A total of 78 sera from donkeys were investigated in Sanliurfa Province, of which 9 (11.5%) were positive by ELISA. Among the 9 positive sera, 6 (12.8%) were from donkeys 1-5 years old and 3 (13.6%) were from donkeys >5 years of age. No positive sera were found in donkeys less than 1 year old. Five (12.5%) sera of females and 4 (10.5%) sera of males tested were positive. These results indicate the existence of R. equi in the horse populations in Sanliurfa and Diyarbakir Provinces. Similar infection rates were found for donkeys in Sanliurfa. This suggests the importance of serological surveys to diagnose R. equi infection in the region and to prevent the zoonotic risk.

  13. Systemic and respiratory oxidative stress in the pathogenesis and diagnosis of Rhodococcus equi pneumonia.

    PubMed

    Crowley, J; Po, E; Celi, P; Muscatello, G

    2013-12-01

    Oxidative stress (OS) is most simply defined as an imbalance between oxidants and antioxidants. Oxidative stress has been suggested to play roles in various equine respiratory diseases and the significance of OS in the pathogenesis of Rhodococcus equi pneumonia is unknown. To measure and relate biomarkers of OS to lesions consistent with R. equi pneumonia. Case-control study. Various OS biomarkers were measured from blood and exhaled breath condensate (EBC) samples collected from 26 foals between 1 and 2 months of age (n = 12 cases and n = 14 controls) on 2 Thoroughbred farms endemically affected by R. equi pneumonia. Foals were defined as cases (positive) or controls (negative) based on ultrasonographic evidence of pulmonary abscessation (>15 mm in diameter). Haematology and biochemistry testing was also performed on blood samples collected from the foals. Comparison of biomarkers and key haematological and biochemical markers of inflammation between the groups was performed using 2 sample t tests. Derivatives of reactive oxygen metabolites (d-ROMs) were significantly greater in case foals than in control foals (P = 0.027) and the oxidative stress index (OSI) was higher in case foals (P = 0.014). Hydrogen peroxide (H2 O2 ) concentrations in EBC were significantly greater in case foals than in control foals (P = 0.002). Meanwhile, there were no significant differences in traditional measures of inflammation between the 2 groups. Measuring OS in both blood and EBC provided useful information in the early diagnosis of R. equi pneumonia. © 2013 EVJ Ltd.

  14. Plasmid Profiles of Virulent Rhodococcus equi Strains Isolated from Infected Foals in Poland.

    PubMed

    Kalinowski, Marcin; Grądzki, Zbigniew; Jarosz, Łukasz; Kato, Kiyoko; Hieda, Yu; Kakuda, Tsutomu; Takai, Shinji

    2016-01-01

    Rhodococcus equi is an important bacterial pathogen in foals up to 6 months old, widespread in horse farms all over the world. It was found that only virulent R. equi strains expressing 15-17 kDa virulence-associated protein (VapA) and having large virulence plasmid of 85-90 kb containing vapA gene are pathogenic for horses. To date, 12 plasmid types have been reported in VapA positive strains from horses. There are no data concerning plasmid types of Polish field R. equi strains isolated from horses and horse farm environment. The aim of the study is to determine plasmid profiles of virulent R. equi strains isolated in Poland from dead foals as well as from soil samples taken from horse breeding farms. Plasmid profiles of 10 clinical strains derived from 8 farms and 11 environmental strains from 3 farms, confirmed as virulent by PCR, were compared with 12 reference strains containing the known plasmid size and type. Plasmid DNAs were analysed by digestion with the restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII for detailed comparison and estimation of plasmid sizes. The results of RFLP analysis revealed that all except one isolates used in the study are classified as VapA 85 kb type I plasmid. One strain harboured VapA 87 kb type I plasmid. This is the first report of plasmid types of Polish field R. equi strains. The results of our preliminary investigations on horse farms located in central and eastern Poland indicate that the virulent R. equi strains thus far isolated from diseased foals and horse farms environment represent a highly uniform plasmid pattern.

  15. IcgA is a virulence factor of Rhodococcus equi that modulates intracellular growth.

    PubMed

    Wang, Xiaoguang; Coulson, Garry B; Miranda-Casoluengo, Aleksandra A; Miranda-Casoluengo, Raúl; Hondalus, Mary K; Meijer, Wim G

    2014-05-01

    Virulence of the intracellular pathogen Rhodococcus equi depends on a 21.3-kb pathogenicity island located on a conjugative plasmid. To date, the only nonregulatory pathogenicity island-encoded virulence factor identified is the cell envelope-associated VapA protein. Although the pathogenicity islands from porcine and equine R. equi isolates have undergone major rearrangements, the virR operon (virR-icgA-vapH-orf7-virS) is highly conserved in both, suggesting these genes play an important role in pathogenicity. VirR and VirS are transcriptional regulators controlling expression of pathogenicity island genes, including vapA. Here, we show that while vapH and orf7 are dispensable for intracellular growth of R. equi, deletion of icgA, formerly known as orf5, encoding a major facilitator superfamily transport protein, elicited an enhanced growth phenotype in macrophages and a significant reduction in macrophage viability, while extracellular growth in broth remained unaffected. Transcription of virS, located downstream of icgA, and vapA was not affected by the icgA deletion during growth in broth or in macrophages, showing that the enhanced growth phenotype caused by deletion of icgA was not mediated through abnormal transcription of these genes. Transcription of icgA increased 6-fold within 2 h following infection of macrophages and remained significantly higher 48 h postinfection compared to levels at the start of the infection. The major facilitator superfamily transport protein IcgA is the first factor identified in R. equi that negatively affects intracellular replication. Aside from VapA, it is only the second pathogenicity island-encoded structural protein shown to play a direct role in intracellular growth of this pathogenic actinomycete.

  16. IcgA Is a Virulence Factor of Rhodococcus equi That Modulates Intracellular Growth

    PubMed Central

    Wang, Xiaoguang; Coulson, Garry B.; Miranda-CasoLuengo, Aleksandra A.; Miranda-CasoLuengo, Raúl; Hondalus, Mary K.

    2014-01-01

    Virulence of the intracellular pathogen Rhodococcus equi depends on a 21.3-kb pathogenicity island located on a conjugative plasmid. To date, the only nonregulatory pathogenicity island-encoded virulence factor identified is the cell envelope-associated VapA protein. Although the pathogenicity islands from porcine and equine R. equi isolates have undergone major rearrangements, the virR operon (virR-icgA-vapH-orf7-virS) is highly conserved in both, suggesting these genes play an important role in pathogenicity. VirR and VirS are transcriptional regulators controlling expression of pathogenicity island genes, including vapA. Here, we show that while vapH and orf7 are dispensable for intracellular growth of R. equi, deletion of icgA, formerly known as orf5, encoding a major facilitator superfamily transport protein, elicited an enhanced growth phenotype in macrophages and a significant reduction in macrophage viability, while extracellular growth in broth remained unaffected. Transcription of virS, located downstream of icgA, and vapA was not affected by the icgA deletion during growth in broth or in macrophages, showing that the enhanced growth phenotype caused by deletion of icgA was not mediated through abnormal transcription of these genes. Transcription of icgA increased 6-fold within 2 h following infection of macrophages and remained significantly higher 48 h postinfection compared to levels at the start of the infection. The major facilitator superfamily transport protein IcgA is the first factor identified in R. equi that negatively affects intracellular replication. Aside from VapA, it is only the second pathogenicity island-encoded structural protein shown to play a direct role in intracellular growth of this pathogenic actinomycete. PMID:24549327

  17. Identification of genomic loci associated with Rhodococcus equi susceptibility in foals.

    PubMed

    McQueen, Cole M; Doan, Ryan; Dindot, Scott V; Bourquin, Jessica R; Zlatev, Zlatomir Z; Chaffin, M Keith; Blodgett, Glenn P; Ivanov, Ivan; Cohen, Noah D

    2014-01-01

    Pneumonia caused by Rhodococcus equi is a common cause of disease and death in foals. Although agent and environmental factors contribute to the incidence of this disease, the genetic factors influencing the clinical outcomes of R. equi pneumonia are ill-defined. Here, we performed independent single nucleotide polymorphism (SNP)- and copy number variant (CNV)-based genome-wide association studies to identify genomic loci associated with R. equi pneumonia in foals. Foals at a large Quarter Horse breeding farm were categorized into 3 groups: 1) foals with R. equi pneumonia (clinical group [N = 43]); 2) foals with ultrasonographic evidence of pulmonary lesions that never developed clinical signs of pneumonia (subclinical group [N = 156]); and, 3) foals without clinical signs or ultrasonographic evidence of pneumonia (unaffected group [N = 49]). From each group, 24 foals were randomly selected and used for independent SNP- and CNV-based genome-wide association studies (GWAS). The SNP-based GWAS identified a region on chromosome 26 that had moderate evidence of association with R. equi pneumonia when comparing clinical and subclinical foals. A joint analysis including all study foals revealed a 3- to 4-fold increase in odds of disease for a homozygous SNP within the associated region when comparing the clinical group with either of the other 2 groups of foals or their combination. The region contains the transient receptor potential cation channel, subfamily M, member 2 (TRPM2) gene, which is involved in neutrophil function. No associations were identified in the CNV-based GWAS. Collectively, these data identify a region on chromosome 26 associated with R. equi pneumonia in foals, providing evidence that genetic factors may indeed contribute to this important disease of foals.

  18. Chloroquine inhibits Rhodococcus equi replication in murine and foal alveolar macrophages by iron-starvation.

    PubMed

    Gressler, Leticia T; Bordin, Angela I; McQueen, Cole M; Cohen, Noah D; de Vargas, Agueda Castagna

    2016-05-30

    Rhodococcus equi preferentially infects macrophages causing pyogranulomatous pneumonia in young foals. Both the vapA and rhbC genes are up-regulated in an iron (Fe)-deprived environment, such as that found within macrophages. Chloroquine (CQ) is a drug widely used against malaria that suppresses the intracellular availability of Fe in eukaryotic cells. The main objective of this study was to evaluate the ability of CQ to inhibit replication of virulent R. equi within murine (J774A.1) and foal alveolar macrophages (AMs) and to verify whether the mechanism of inhibition could be Fe-deprivation-dependent. CQ effect on R. equi extracellular survival and toxicity to J774A.1 were evaluated. R. equi survival within J774A.1 and foal AMs was evaluated under CQ (10 and 20μM), bovine saturated transferrin (bHTF), and bovine unsaturated transferrin (bATF) exposure. To explore the action mechanism of CQ, the superoxide anion production, the lysozyme activity, as well as the relative mRNA expression of vapA and rhbC were examined. CQ at≤20μM had no effect on R. equi extracellular multiplication and J774A.1 viability. Exposure to CQ significantly and markedly reduced survival of R. equi within J774A.1 and foal AMs. Treatment with bHTF did not reverse CQ effect on R. equi. Exposure to CQ did not affected superoxide anion production or lysozyme activity, however vapA and rhbC expression was significantly increased. Our results reinforce the hypothesis that intracellular availability of Fe is required for R. equi survival, and our initial hypothesis that CQ can limit replication of R. equi in J774A.1 and foal AMs, most likely by Fe starvation. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Plasmid Profiles of Virulent Rhodococcus equi Strains Isolated from Infected Foals in Poland

    PubMed Central

    Kalinowski, Marcin; Grądzki, Zbigniew; Jarosz, Łukasz; Kato, Kiyoko; Hieda, Yu; Kakuda, Tsutomu; Takai, Shinji

    2016-01-01

    Rhodococcus equi is an important bacterial pathogen in foals up to 6 months old, widespread in horse farms all over the world. It was found that only virulent R. equi strains expressing 15–17 kDa virulence-associated protein (VapA) and having large virulence plasmid of 85–90 kb containing vapA gene are pathogenic for horses. To date, 12 plasmid types have been reported in VapA positive strains from horses. There are no data concerning plasmid types of Polish field R. equi strains isolated from horses and horse farm environment. The aim of the study is to determine plasmid profiles of virulent R. equi strains isolated in Poland from dead foals as well as from soil samples taken from horse breeding farms. Plasmid profiles of 10 clinical strains derived from 8 farms and 11 environmental strains from 3 farms, confirmed as virulent by PCR, were compared with 12 reference strains containing the known plasmid size and type. Plasmid DNAs were analysed by digestion with the restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII for detailed comparison and estimation of plasmid sizes. The results of RFLP analysis revealed that all except one isolates used in the study are classified as VapA 85 kb type I plasmid. One strain harboured VapA 87 kb type I plasmid. This is the first report of plasmid types of Polish field R. equi strains. The results of our preliminary investigations on horse farms located in central and eastern Poland indicate that the virulent R. equi strains thus far isolated from diseased foals and horse farms environment represent a highly uniform plasmid pattern. PMID:27074033

  20. Air sampling in the breathing zone of neonatal foals for prediction of subclinical Rhodococcus equi infection.

    PubMed

    Chicken, C; Muscatello, G; Freestone, J; Anderson, G A; Browning, G F; Gilkerson, J R

    2012-03-01

    Disease caused by Rhodococcus equi is a significant burden to the horse breeding industry worldwide. Early detection of rhodococcal pneumonia, albeit important to minimise treatment costs, is difficult because of the insidious nature of the disease and the lack of definitive diagnostic tests. To investigate air sampling from the breathing zone of neonatal foals as a predictor of subsequent rhodococcal pneumonia. Air samples were collected from the breathing zone of 53 neonatal foals (age ≤10 days) and again at the time of routine ultrasonographic screening for R. equi pneumonia (age 1-2 months). Pneumonia was diagnosed ultrasonographically in 23% of foals. Virulent R. equi was detected in air from the breathing zone of 19% of neonatal foals and 45% of foals at age 1-2 months. There was no association between virulent R. equi in the breathing zone of foals and the subsequent ultrasonographic diagnosis of rhodococcal pneumonia. The median concentration of virulent R. equi in the breathing zone of both neonates (0 [range 0-4] colony-forming units [cfu]/250 l) and older foals (0 [range 0-3] cfu/250 l) was not significantly different from that in background air samples (0 [range 0-6] cfu/250 l). There was no difference in the concentration of virulent R. equi in the breathing zone of older foals that were diagnosed with rhodococcal pneumonia or clinically normal foals. Detection of virulent R. equi in air from the breathing zone was not a positive predictor of rhodococcal pneumonia in foals up to age ≤2 months. Selective culture of air samples from the breathing zone of young foals is not better at diagnosing rhodococcal pneumonia than early ultrasonographic screening. However, culture of air samples from the breathing zone of older foals remains a useful herd-based epidemiological tool. © 2012 EVJ Ltd.

  1. Novel transferable erm(46) determinant responsible for emerging macrolide resistance in Rhodococcus equi.

    PubMed

    Anastasi, Elisa; Giguère, Steeve; Berghaus, Londa J; Hondalus, Mary K; Willingham-Lane, Jennifer M; MacArthur, Iain; Cohen, Noah D; Roberts, Marilyn C; Vazquez-Boland, Jose A

    2015-12-01

    The objective of this study was to identify the molecular mechanism of macrolide resistance in the actinomycete Rhodococcus equi, a major equine pathogen and zoonotic agent causing opportunistic infections in people. Macrolide-resistant (n = 62) and macrolide-susceptible (n = 62) clinical isolates of R. equi from foals in the USA were studied. WGS of 18 macrolide-resistant and 6 macrolide-susceptible R. equi was performed. Representative sequences of all known macrolide resistance genes identified to date were used to search the genome assemblies for putative homologues. PCR was used to screen for the presence of the identified resistance determinant in the rest of the isolates. Mating experiments were performed to verify mobility of the gene. A novel erm gene, erm(46), was identified in all sequenced resistant isolates, but not in susceptible isolates. There was complete association between macrolide resistance and the presence of erm(46) as detected by PCR screening of all 124 clinical isolates of R. equi. Expression of erm(46) in a macrolide-susceptible strain of R. equi induced high-level resistance to macrolides, lincosamides and streptogramins B, but not to other classes of antimicrobial agents. Transfer of erm(46) to macrolide-susceptible R. equi was confirmed. The transfer frequency ranged from 3 × 10(-3) to 1 × 10(-2). This is the first molecular characterization of resistance to macrolides, lincosamides and streptogramins B in R. equi. Resistance was due to the presence of a novel erm(46) gene mobilizable likely by conjugation, which has spread among equine isolates of R. equi in the USA. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  2. Mutant prevention concentration and mutant selection window for 10 antimicrobial agents against Rhodococcus equi.

    PubMed

    Berghaus, Londa J; Giguère, Steeve; Guldbech, Kristen

    2013-10-25

    The objectives of this study were to determine the mutant prevention concentration (MPC), time above the MPC and mutant selection window for 10 antimicrobial agents against Rhodococcus equi and to determine if the combination of a macrolide with rifampin would decrease emergence of resistant mutants. Antimicrobial agents investigated (erythromycin, clarithromycin, azithromycin, rifampin, amikacin, gentamicin, enrofloxacin, vancomycin, imipenem, and doxycycline) were selected based on in vitro activity and frequency of use in foals or people infected with R. equi. Each antimicrobial agent or combination of agents was evaluated against four virulent strains of R. equi. MPC were determined using an agar plate assay. Pharmacodynamic parameters were calculated using published plasma and pulmonary pharmacokinetic variables. There was a significant (P<0.001) effect of the type of antimicrobial agent on the MPC. The MPC of clarithromycin (1.0 μg/ml) was significantly lower and the MPC of rifampin and amikacin (512 and 384 μg/ml, respectively) were significantly higher than that of all other antimicrobial agents tested. Combining erythromycin, clarithromycin, or azithromycin with rifampin resulted in a significant (P≤0.005) decrease in MPC and MPC/MIC ratio. When MIC and MPC were combined with pharmacokinetic variables, only gentamicin and vancomycin were predicted to achieve plasma concentrations above the MPC for any given periods of time. Only clarithromycin and the combination clarithromycin-rifampin were predicted to achieve concentrations in bronchoalveolar cells and pulmonary epithelial lining fluid above the MPC for the entire dosing interval. In conclusion, the combination of a macrolide with rifampin considerably decreases the emergence of resistant mutants of R. equi. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Effects of opsonization of Rhodococcus equi on bacterial viability and phagocyte activation.

    PubMed

    Dawson, Dominic R; Nydam, Daryl V; Price, Christopher T; Graham, James E; Cynamon, Michael H; Divers, Thomas J; Felippe, Maria Julia B

    2011-11-01

    To investigate the effect of opsonization of Rhodococcus equi with R. equi-specific antibodies in plasma on bacterial viability and phagocyte activation in a cell culture model of infection. Neutrophils and monocyte-derived macrophages from 6 healthy 1-week-old foals and 1 adult horse. Foal and adult horse phagocytes were incubated with either opsonized or nonopsonized bacteria. Opsonization was achieved by use of plasma containing high or low concentrations of R. equi-specific antibodies. Phagocyte oxidative burst activity was measured by use of flow cytometry, and macrophage tumor necrosis factor (TNF)-α production was measured via an ELISA. Extracellular and intracellular bacterial viability was measured with a novel R. equi-luciferase construct that used a luminometer. Opsonized bacteria increased oxidative burst activity in adult horse phagocytes, and neutrophil activity was dependent on the concentration of specific antibody. Secretion of TNF-α was higher in macrophages infected with opsonized bacteria. Opsonization had no significant effect on bacterial viability in macrophages; however, extracellular bacterial viability was decreased in broth containing plasma with R. equi-specific antibodies, compared with viability in broth alone. The use of plasma enriched with specific antibodies for the opsonization of R. equi increased the activation of phagocytes and decreased bacterial viability in the extracellular space. Although opsonized R. equi increased TNF-α secretion and oxidative burst in macrophages, additional factors may be necessary for effective intracellular bacterial killing. These data have suggested a possible role of plasma antibody in protection of foals from R. equi pneumonia.

  4. Gallium maltolate as an alternative to macrolides for treatment of presumed Rhodococcus equi pneumonia in foals.

    PubMed

    Cohen, Noah D; Slovis, Nathan M; Giguère, Steeve; Baker, Samantha; Chaffin, M Keith; Bernstein, Lawrence R

    2015-01-01

    Macrolide-resistant isolates of Rhodococcus equi are emerging, prompting the search for clinically effective alternative antimicrobials. The proportion of foals with ultrasonographic evidence of pneumonia presumed to be caused by R. equi that had a successful outcome when administered gallium maltolate (GaM) PO would not be more than 10% inferior (ie, lower) than that of foals receiving standard treatment. Fifty-four foals with subclinical pulmonary abscesses among 509 foals at 6 breeding farms in Kentucky. Controlled, randomized, prospective noninferiority study. Foals with ultrasonographic lesions >1 cm in diameter (n = 54) were randomly allocated to receive per os either clarithromycin combined with rifampin (CLR+R) or GaM, and followed up for 28 days by daily physical inspections and weekly (n = 1 farm) or biweekly (n = 4 farms) thoracic ultrasound examinations by individuals unaware of treatment-group assignments. Treatment success was defined as resolution of ultrasonographically identified pulmonary abscesses within 28 days of initiating treatment. Noninferiority was defined as a 90% confidence interval for the observed difference in CLR+R minus GaM that was ≤10%. The proportion of GaM-treated foals that resolved (70%; 14/20) was similar to that of foals treated with CLR+R (74%; 25/34), but we failed to demonstrate noninferiority for GaM relative to CLR+R; however, GaM was noninferior to CLR+R treatment when results from a noncompliant farm were excluded. Gallium maltolate is not inferior to macrolides for treating foals with subclinical pneumonia. Use of GaM might reduce pressure for macrolide-resistance in R. equi. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

  5. The effect of bacterial dose and foal age at challenge on Rhodococcus equi infection.

    PubMed

    Sanz, M; Loynachan, A; Sun, L; Oliveira, A; Breheny, P; Horohov, D W

    2013-12-27

    While Rhodococcus equi remains the most common cause of subacute or chronic granulomatous bronchopneumonia in foals, development of a relevant model to study R. equi infection has proven difficult. The objective of this study was to identify a challenge dose of R. equi that resulted in slow progressive disease, spontaneous regression of lung lesions and age-dependent susceptibility. Foals less than one-week of age were challenged intratracheally using either 10(6), 10(5), 10(4), 10(3) or 10(2) cfu of R. equi. Two doses (10(3) cfu and 10(5) cfu) were used to challenge 2 and 3-week-old, and 3 and 6-week-old foals, respectively. Physical examination, thoracic ultrasound and blood work were performed. Foals were euthanized at the end of the study or when clinical signs of pneumonia developed. All foals were necropsied and their lung lesions scored. Foals challenged with low concentrations of R. equi developed slow progressive pneumonia and approximately 50% of the foals recovered spontaneously. Likewise, macroscopic (>1cm diameter) pyogranulomatous lesions were only observed when low doses of R. equi were used. Clinical pneumonia was not seen after low dose challenge in the 3-week-old foals or in the 6-week-old foals. This study demonstrates that the use of low doses of R. equi to challenge neonatal foals provides an improved model for studying this disease. Furthermore, susceptibility to R. equi infection was shown to diminish early in the foal's life, as has been reported in the field. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Relationship of mixed bacterial infection to prognosis in foals with pneumonia caused by Rhodococcus equi.

    PubMed

    Giguère, S; Jordan, L M I; Glass, K; Cohen, N D

    2012-01-01

    Isolation of multiple bacterial species is common in foals with Rhodococcus equi pneumonia. There is no association between isolation of other microorganisms and outcome. 155 foals with pneumonia caused by R. equi. Case records of foals diagnosed with R. equi pneumonia based on culture of the respiratory tract were reviewed at 2 referral hospitals (University of Florida [UF] and Texas A&M University [TAMU]). R. equi was cultured from a tracheobronchial aspirate (TBA) in 115 foals and from lung tissue in 38 foals. Survival was significantly higher at UF (71%; 70/99) than at TAMU (50%; 28/56). R. equi was significantly more likely to grow in pure cultures from samples obtained from foals at UF (55%; 54/99) than from foals at TAMU (23%; 13/56). Microorganisms cultured with R. equi included Gram-positive bacteria in 40, Gram-negative bacteria in 41, and fungi in 23 foals. The most common bacteria isolated were beta-hemolytic streptococci (n = 26) and Escherichia coli (n = 18). Mixed infections were significantly more likely to be encountered in TBA than in lung tissue. Only foals from which R. equi was cultured from a TBA were included in the analysis for association between mixed infection and outcome. After adjusting for the effect of hospital using multivariate logistic regression, mixed culture, mixed bacterial culture, Gram-positive bacteria, beta-hemolytic streptococci, Gram-negative bacteria, enteric Gram-negative bacteria, nonenteric Gram-negative bacteria, and fungi were not significantly associated with outcome. Isolation of multiple bacteria or fungi from a TBA along with R. equi does not negatively impact prognosis. Copyright © 2012 by the American College of Veterinary Internal Medicine.

  7. Mouse lung infection model to assess Rhodococcus equi virulence and vaccine protection.

    PubMed

    González-Iglesias, Patricia; Scortti, Mariela; MacArthur, Iain; Hapeshi, Alexia; Rodriguez, Héctor; Prescott, John F; Vazquez-Boland, José A

    2014-08-06

    The pathogenic actinomycete Rhodococcus equi causes severe purulent lung infections in foals and immunocompromised people. Although relatively unsusceptible to R. equi, mice are widely used for in vivo studies with this pathogen. The most commonly employed mouse model is based on systemic (intravenous) infection and determination of R. equi burdens in spleen and liver. Here, we investigated the murine lung for experimental infection studies with R. equi. Using a 10(7)CFU intranasal challenge in BALB/c mice, virulent R. equi consistently survived in quantifiable numbers up to 10 days in the lungs whereas virulence-deficient R. equi bacteria were rapidly cleared. An internally controlled virulence assay was developed in which the test R. equi strains are co-inoculated and monitored in the same mouse. Isogenic R. equi bacteria lacking either the plasmid vapA gene or the entire virulence plasmid were compared using this competitive assay. Both strains showed no significant differences in in vivo fitness in the lung, indicating that the single loss of the virulence factor VapA was sufficient to account for the full attenuation seen in the absence of the virulence plasmid. To test the adequacy of the lung infection model for monitoring R. equi vaccine efficacy, BALB/c mice were immunized with live R. equi and challenged intranasally. Vaccination conferred protection against acute pulmonary challenge with virulent R. equi. Our data indicate that the murine lung infection model provides a useful tool for both R. equi virulence and vaccine studies. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Use of Liposomal Gentamicin for Treatment of 5 Foals with Experimentally Induced Rhodococcus equi Pneumonia.

    PubMed

    Cohen, N D; Giguère, S; Burton, A J; Rocha, J N; Berghaus, L J; Brake, C N; Bordin, A I; Coleman, M C

    2016-01-01

    Adverse effects of, and bacterial resistance to, macrolides used to treat Rhodococcus equi infections have prompted search for clinically effective alternative antimicrobials. Liposomal gentamicin (LG) is effective against R. equi in vitro and decreases tissue concentrations of R. equi in experimentally infected mice. Effectiveness of LG treatment of foals with R. equi pneumonia, however, has not been described. Liposomal gentamicin is safe and effective for treating foals with R. equi pneumonia. Ten foals with experimentally induced R. equi pneumonia. Pilot treatment trial. Foals with pneumonia induced by intrabronchial instillation of R. equi were randomly allocated to receive either clarithromycin combined with rifampin (CLR + RIF) PO or LG IV, and followed by daily physical examinations and weekly thoracic ultrasonography and serum creatinine concentration determinations until the resolution of clinical signs. Treatment success was defined as the resolution of clinical signs and ultrasonographically identified pulmonary abscesses. All 10 foals were successfully treated. Two of 5 foals treated with LG developed azotemia within 1 week; LG was discontinued and treatment switched to CLR + RIF for these foals. None of the CLR + RIF treated foals developed azotemia. Liposomal gentamicin IV can be effective for treatment of R. equi pneumonia, but nephrotoxicity indicates that an alternative dosing interval or route (such as nebulization) will be needed before LG is adequately safe for clinical use. Larger comparative trials will be needed to evaluate the relative efficacy of a safer LG dosage regimen. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

  9. In vitro synergy, pharmacodynamics, and postantibiotic effect of 11 antimicrobial agents against Rhodococcus equi.

    PubMed

    Giguère, Steeve; Lee, Elise A; Guldbech, Kristen M; Berghaus, Londa J

    2012-11-09

    There are no studies investigating interactions between clarithromycin or azithromycin and rifampin or other commonly used antimicrobial agents against virulent isolates of Rhodococcus equi. In addition, there is no published data on the postantibiotic effects (PAEs) and pharmacodynamics properties of antimicrobial agents against R. equi. The objectives were to assess in vitro interactions, pharmacodynamics, and PAEs of 11 antimicrobial agents belonging to various antimicrobial classes against R. equi. Antimicrobial agents investigated (erythromycin, clarithromycin, azithromycin, rifampin, amikacin, gentamicin, enrofloxacin, vancomycin, imipenem, ceftiofur, and doxycycline) were selected based on in vitro activity against large numbers of isolates of R. equi and frequency of use in foals or humans infected with R. equi. Three virulent strains of R. equi were evaluated by time-kill curves and checkerboard assays, and the postantibiotic effect was measured at 5×MIC. Only amikacin, gentamicin, enrofloxacin, and vancomycin were bactericidal against R. equi. Combinations including a macrolide (erythromycin, clarithromycin, azithromycin) and either rifampin or doxycycline, and the combination doxycycline-rifampin were synergistic. Combinations containing amikacin and erythromycin, clarithromycin, azithromycin, or rifampin and the combination gentamicin-rifampin were antagonistic. The PAEs of rifampin, erythromycin, clarithromycin, vancomycin, and doxycycline were relatively long with median values ranging between 4.5 and 6.5h. Azithromycin, gentamicin, and imipenem had intermediate PAEs ranging between 3.3 and 3.5h. Amikacin, enrofloxacin, and ceftiofur had shorter PAEs ranging between 1.3 and 2.1h. Gentamicin, amikacin, enrofloxacin, and doxycycline exhibited concentration-dependent activity whereas erythromycin, clarithromycin, azithromycin, rifampin, ceftiofur, imipenem, and vancomycin exhibited time-dependent activity against R. equi. Copyright © 2012 Elsevier B

  10. MICs of 32 antimicrobial agents for Rhodococcus equi isolates of animal origin.

    PubMed

    Riesenberg, Anne; Feßler, Andrea T; Erol, Erdal; Prenger-Berninghoff, Ellen; Stamm, Ivonne; Böse, Reinhard; Heusinger, Anton; Klarmann, Dieter; Werckenthin, Christiane; Schwarz, Stefan

    2014-04-01

    The aim of this study was to determine the MICs of 32 antimicrobial agents for 200 isolates of Rhodococcus equi of animal origin by applying a recently described broth microdilution protocol, and to investigate isolates with distinctly elevated rifampicin MICs for the genetic basis of rifampicin resistance. The study included 200 R. equi isolates, including 160 isolates from horses and 40 isolates from other animal sources, from the USA and Europe. MIC testing of 32 antimicrobial agents or combinations thereof followed a recently published protocol. A novel PCR protocol for the joint amplification of the three rpoB regions in which rifampicin resistance-mediating mutations have been reported was applied to isolates with elevated rifampicin MICs. The amplicons were sequenced and screened for mutations. Susceptibility testing revealed a rather uniform distribution of MICs for most of the antimicrobial agents tested. The lowest MICs were seen for clarithromycin, rifampicin and imipenem. Six isolates (3%) exhibited distinctly higher MICs of rifampicin than the remaining 194 isolates. In five of these six isolates, single bp exchanges, which resulted in the amino acid exchanges Gln513Leu, Asp516Val, His526Asp or Ser531Leu, were detected in the rifampicin resistance-determining region 1 of the rpoB gene, with Gln513Leu representing a novel substitution for R. equi. This study shows the MIC distribution of 32 antimicrobial agents for a large collection of R. equi isolates of animal origin from two continents. Isolates that exhibited distinctly elevated MICs of rifampicin were only rarely detected.

  11. Isolation and Characterization of Carbendazim-degrading Rhodococcus erythropolis djl-11

    PubMed Central

    Harvey, Paul R.; Li, Hongmei; Ren, Yan; Li, Jishun; Wang, Jianing; Yang, Hetong

    2013-01-01

    Carbendazim (methyl 1H-benzimidazol-2-yl carbamate) is one of the most widely used fungicides in agriculture worldwide, but has been reported to have adverse effects on animal health and ecosystem function. A highly efficient carbendazim-degrading bacterium (strain dj1-11) was isolated from carbendazim-contaminated soil samples via enrichment culture. Strain dj1-11 was identified as Rhodococcus erythropolis based on morphological, physiological and biochemical characters, including sequence analysis of the 16S rRNA gene. In vitro degradation of carbendazim (1000 mg·L−1) by dj1-11 in minimal salts medium (MSM) was highly efficient, and with an average degradation rate of 333.33 mg·L−1·d−1 at 28°C. The optimal temperature range for carbendazim degradation by dj1-11 in MSM was 25–30°C. Whilst strain dj1-11 was capable of metabolizing cabendazim as the sole source of carbon and nitrogen, degradation was significantly (P<0.05) increased by addition of 12.5 mM NH4NO3. Changes in MSM pH (4–9), substitution of NH4NO3 with organic substrates as N and C sources or replacing Mg2+ with Mn2+, Zn2+ or Fe2+ did not significantly affect carbendazim degradation by dj1-11. During the degradation process, liquid chromatography-mass spectrometry (LC-MS) detected the metabolites 2-aminobenzimidazole and 2-hydroxybenzimidazole. A putative carbendazim-hydrolyzing esterase gene was cloned from chromosomal DNA of djl-11 and showed 99% sequence homology to the mheI carbendazim-hydrolyzing esterase gene from Nocardioides sp. SG-4G. PMID:24098350

  12. Rhodococcus erythropolis BG43 Genes Mediating Pseudomonas aeruginosa Quinolone Signal Degradation and Virulence Factor Attenuation.

    PubMed

    Müller, Christine; Birmes, Franziska S; Rückert, Christian; Kalinowski, Jörn; Fetzner, Susanne

    2015-11-01

    Rhodococcus erythropolis BG43 is able to degrade the Pseudomonas aeruginosa quorum sensing signal molecules PQS (Pseudomonas quinolone signal) [2-heptyl-3-hydroxy-4(1H)-quinolone] and HHQ [2-heptyl-4(1H)-quinolone] to anthranilic acid. Based on the hypothesis that degradation of HHQ might involve hydroxylation to PQS followed by dioxygenolytic cleavage of the heterocyclic ring and hydrolysis of the resulting N-octanoylanthranilate, the genome was searched for corresponding candidate genes. Two gene clusters, aqdA1B1C1 and aqdA2B2C2, each predicted to code for a hydrolase, a flavin monooxygenase, and a dioxygenase related to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, were identified on circular plasmid pRLCBG43 of strain BG43. Transcription of all genes was upregulated by PQS, suggesting that both gene clusters code for alkylquinolone-specific catabolic enzymes. An aqdR gene encoding a putative transcriptional regulator, which was also inducible by PQS, is located adjacent to the aqdA2B2C2 cluster. Expression of aqdA2B2C2 in Escherichia coli conferred the ability to degrade HHQ and PQS to anthranilic acid; however, for E. coli transformed with aqdA1B1C1, only PQS degradation was observed. Purification of the recombinant AqdC1 protein verified that it catalyzes the cleavage of PQS to form N-octanoylanthranilic acid and carbon monoxide and revealed apparent Km and kcat values for PQS of ∼27 μM and 21 s(-1), respectively. Heterologous expression of the PQS dioxygenase gene aqdC1 or aqdC2 in P. aeruginosa PAO1 quenched the production of the virulence factors pyocyanin and rhamnolipid and reduced the synthesis of the siderophore pyoverdine. Thus, the toolbox of quorum-quenching enzymes is expanded by new PQS dioxygenases.

  13. Rhodococcus erythropolis BG43 Genes Mediating Pseudomonas aeruginosa Quinolone Signal Degradation and Virulence Factor Attenuation

    PubMed Central

    Müller, Christine; Birmes, Franziska S.; Rückert, Christian; Kalinowski, Jörn

    2015-01-01

    Rhodococcus erythropolis BG43 is able to degrade the Pseudomonas aeruginosa quorum sensing signal molecules PQS (Pseudomonas quinolone signal) [2-heptyl-3-hydroxy-4(1H)-quinolone] and HHQ [2-heptyl-4(1H)-quinolone] to anthranilic acid. Based on the hypothesis that degradation of HHQ might involve hydroxylation to PQS followed by dioxygenolytic cleavage of the heterocyclic ring and hydrolysis of the resulting N-octanoylanthranilate, the genome was searched for corresponding candidate genes. Two gene clusters, aqdA1B1C1 and aqdA2B2C2, each predicted to code for a hydrolase, a flavin monooxygenase, and a dioxygenase related to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, were identified on circular plasmid pRLCBG43 of strain BG43. Transcription of all genes was upregulated by PQS, suggesting that both gene clusters code for alkylquinolone-specific catabolic enzymes. An aqdR gene encoding a putative transcriptional regulator, which was also inducible by PQS, is located adjacent to the aqdA2B2C2 cluster. Expression of aqdA2B2C2 in Escherichia coli conferred the ability to degrade HHQ and PQS to anthranilic acid; however, for E. coli transformed with aqdA1B1C1, only PQS degradation was observed. Purification of the recombinant AqdC1 protein verified that it catalyzes the cleavage of PQS to form N-octanoylanthranilic acid and carbon monoxide and revealed apparent Km and kcat values for PQS of ∼27 μM and 21 s−1, respectively. Heterologous expression of the PQS dioxygenase gene aqdC1 or aqdC2 in P. aeruginosa PAO1 quenched the production of the virulence factors pyocyanin and rhamnolipid and reduced the synthesis of the siderophore pyoverdine. Thus, the toolbox of quorum-quenching enzymes is expanded by new PQS dioxygenases. PMID:26319870

  14. Molecular epidemiology of Rhodococcus equi in slaughtered swine, cattle and horses in Poland.

    PubMed

    Witkowski, Lucjan; Rzewuska, Magdalena; Takai, Shinji; Kizerwetter-Świda, Magdalena; Kita, Jerzy

    2016-05-27

    Rhodococcus equi is an emerging zoonotic presumably foodborne pathogen. Since the data on the worldwide prevalence of R. equi in meat animals are scarce, the present study aimed to investigate the molecular epidemiology of R. equi in swine, cattle and horse carcasses intended for human consumption in Poland. Totally 1028 lymph node samples were examined. R. equi was isolated from 26.6 % (105/395) swine and 1.3 % (3/234) bovine healthy submaxillary lymph nodes. In horses, R. equi was isolated only from 0.5 % (1/198) samples of middle tracheo-branchiales lymph node while no lymphocentrum retropharyngeum sample was positive (0/198). The purulent lesions were observed only in 0.8 % swine submaxillary lymph nodes samples (3/398) and in two of them R. equi was detected. All bovine and most of swine isolates (98.1 %) were vapB-positive. 87.9 % of swine isolates carried 95-kb type 5 plasmid, 3.7 % type 1 and plasmid types: 4, 7, 10, 11, 21, 31 were carried by a single isolate (0.9 %). All bovine isolates carried VAPB type 26. Single horse isolate was vapA-positive and carried plasmid VAPA 85-kb type I. The prevalence of vapB-positive R. equi in investigated healthy swine intended for human consumption was very high. Not only swine, but also even apparently healthy cattle or horse carcasses should be considered as a potential source of R. equi for humans, especially in countries where undercooked or raw beef or horsemeat is traditionally consumed.

  15. Insight into Cr(6+) reduction efficiency of Rhodococcus erythropolis isolated from coalmine waste water.

    PubMed

    Banerjee, Soumya; Joshi, S R; Mandal, Tamal; Halder, Gopinath

    2017-01-01

    A microbial treatment of Cr(6+) contaminated wastewater with a chromium reducing bacteria isolated from coal mine area was investigated. In a series of batch study metal removal was executed under different parametric conditions which include pH (2-7), temperature (20-50 °C), initial Cr(6+) concentration (1-100 mg/L), substrate utilization and its overall effect on biomass generation. Impact of oxygen availability was checked at different agitation speed and its role on the remedial process. Liquid phase reduction of Cr(6+) was measured in terms of substrate reduction and total biomass yield. The bacterium species isolated was able to tolerate Cr(6+) over a wide range from 1 to 100 mg/L before it reached minimum inhibition concentration. Apart from Cr(6+), the bacterial isolate showed tolerance towards Fe, As, Cu, Ag, Zn, Mn, Mg and Pb. Removal mechanism adopted by the bacterium recommended that it employed accumulation of Cr(6+) as Cr(3+) both within and outside the cell. Classical Monod equation was used to determine the biokinetics of the bacterial isolate along with the interference of metal ion concentration and substrate utilization. Cr(6+) removal was found prominent even in bimetallic solutions. The bacterial isolate was confirmed to be Rhodococcus erythopolis by 16s rRNA molecular characterization. Thus the bacterial isolate obtained from the coal mine area proved to be a potential agent for microbial remediation of Cr(6+) laden waste water. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Evidence for an Inducible Nucleotide-Dependent Acetone Carboxylase in Rhodococcus rhodochrous B276

    PubMed Central

    Clark, Daniel D.; Ensign, Scott A.

    1999-01-01

    The metabolism of acetone was investigated in the actinomycete Rhodococcus rhodochrous (formerly Nocardia corallina) B276. Suspensions of acetone- and isopropanol-grown R. rhodochrous readily metabolized acetone. In contrast, R. rhodochrous cells cultured with glucose as the carbon source lacked the ability to metabolize acetone at the onset of the assay but gained the ability to do so in a time-dependent fashion. Chloramphenicol and rifampin prevented the time-dependent increase in this activity. Acetone metabolism by R. rhodochrous was CO2 dependent, and 14CO2 fixation occurred concomitant with this process. A nucleotide-dependent acetone carboxylase was partially purified from cell extracts of acetone-grown R. rhodochrous by DEAE-Sepharose chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the acetone carboxylase was composed of three subunits with apparent molecular masses of 85, 74, and 16 kDa. Acetone metabolism by the partially purified enzyme was dependent on the presence of a divalent metal and a nucleoside triphosphate. GTP and ITP supported the highest rates of acetone carboxylation, while CTP, UTP, and XTP supported carboxylation at 10 to 50% of these rates. ATP did not support acetone carboxylation. Acetoacetate was determined to be the stoichiometric product of acetone carboxylation. The longer-chain ketones butanone, 2-pentanone, 3-pentanone, and 2-hexanone were substrates. This work has identified an acetone carboxylase with a novel nucleotide usage and broader substrate specificity compared to other such enzymes studied to date. These results strengthen the proposal that carboxylation is a common strategy used for acetone catabolism in aerobic acetone-oxidizing bacteria. PMID:10217764

  17. Laboratory-scale biofiltration of acrylonitrile by Rhodococcus rhodochrous DAP 96622 in a trickling bed bioreactor.

    PubMed

    Zhang, Jie; Pierce, George E

    2009-07-01

    Acrylonitrile (ACN), a volatile component of the waste generated during the production of acrylamide, also is often associated with aromatic contaminants such as toluene and styrene. Biofiltration, considered an effective technique for the treatment of volatile hydrocarbons, has not been used to treat volatile nitriles. An experimental laboratory-scale trickling bed bioreactor using cells of Rhodococcus rhodochrous DAP 96622 supported on granular activated carbon (GAC) was developed and evaluated to assess the ability of biofiltration to treat ACN. In addition to following the course of treatability of ACN, kinetics of ACN biodegradation during both recycle batch and open modes of operation by immobilized and free cells were evaluated. For fed-batch mode bioreactor with immobilized cells, almost complete ACN removal (>95%) was achieved at a flow rate of 0.1 microl/min ACN and 0.8 microl/min toluene (TOL) (for comparative purposes this is equivalent to 6.9 mg l(-1) h(-1) ACN and 83.52 mg l(-1) h(-1) TOL). In a single-pass mode bioreactor with immobilized cells, at ACN inlet loads of 100-200 mg l(-1) h(-1) and TOL inlet load of approximately 400 mg l(-1) h(-1), with empty bed retention time (EBRT) of 8 min, ACN removal efficiency was approximately 90%. The three-dimensional structure and characteristics of the biofilm were investigated using confocal scanning laser microscopy (CSLM). CLSM images revealed a robust and heterogeneous biofilm, with microcolonies interspersed with voids and channels. Analysis of the precise measurement of biofilm characteristics using COMSTAT agreed with the assumption that both biomass and biofilm thickness increased along the carbon column depth.

  18. Adsorption of Rhodococcus Strain GIN-1 (NCIMB 40340) on Titanium Dioxide and Coal Fly Ash Particles

    PubMed Central

    Shabtai, Y.; Fleminger, G.

    1994-01-01

    Rhodococcus strain GIN-1 (NCIMB 40340) can be used to enrich and isolate a titanium-rich fraction from coal fly ash. The gram-positive bacterium was isolated by its ability to adhere strongly and rapidly to suspended particles of pure titanium dioxide or coal fly ash. Adsorption depends on the salt concentration and occurs in seawater. Lowering of the salt concentration or washing of particles with pure water did not, however, cause desorption of the bacteria from TiO2 particles; this was achieved by strong alkaline treatment or combined treatment with sodium dodecyl sulfate and urea but not with dilute acids, alcohols, or cationic or nonionic detergents. The bacterium exhibits higher affinity towards oxides of Ti and Zn than to other oxides with similar distribution of particle size. Moreover, it adheres much faster to TiO2 than to magnetite (Fe3O4) or Al2O3. After about 1 min, more than 85% of the cells were adsorbed on TiO2, compared with adsorption of only 10 and 8% to magnetite and Al2O3, respectively. Adsorption of the bacteria on TiO2 occurs over a pH range of 1.0 to 9.0 and at temperatures from 4 to over 80°C. Scanning electron microscopy combined with X-ray analysis revealed preferential adherence of the bacterium to coal ash particles richer in Ti. Stronger adhesion to TiO2 was also demonstrated in the translocation of bacteria, preadsorbed on magnetite, to TiO2 particles. The temporary co-adhesion to magnetite and TiO2 was exploited for the design of a prototype biomagnetic separation process in which bacterial cells serve as an adhesive mediator between magnetite and TiO2 particles in a mixture of Al, Si, and Ti oxides that simulates their proportion in the ash. Images PMID:16349369

  19. Adsorption of Rhodococcus Strain GIN-1 (NCIMB 40340) on Titanium Dioxide and Coal Fly Ash Particles.

    PubMed

    Shabtai, Y; Fleminger, G

    1994-09-01

    Rhodococcus strain GIN-1 (NCIMB 40340) can be used to enrich and isolate a titanium-rich fraction from coal fly ash. The gram-positive bacterium was isolated by its ability to adhere strongly and rapidly to suspended particles of pure titanium dioxide or coal fly ash. Adsorption depends on the salt concentration and occurs in seawater. Lowering of the salt concentration or washing of particles with pure water did not, however, cause desorption of the bacteria from TiO(2) particles; this was achieved by strong alkaline treatment or combined treatment with sodium dodecyl sulfate and urea but not with dilute acids, alcohols, or cationic or nonionic detergents. The bacterium exhibits higher affinity towards oxides of Ti and Zn than to other oxides with similar distribution of particle size. Moreover, it adheres much faster to TiO(2) than to magnetite (Fe(3)O(4)) or Al(2)O(3). After about 1 min, more than 85% of the cells were adsorbed on TiO(2), compared with adsorption of only 10 and 8% to magnetite and Al(2)O(3), respectively. Adsorption of the bacteria on TiO(2) occurs over a pH range of 1.0 to 9.0 and at temperatures from 4 to over 80 degrees C. Scanning electron microscopy combined with X-ray analysis revealed preferential adherence of the bacterium to coal ash particles richer in Ti. Stronger adhesion to TiO(2) was also demonstrated in the translocation of bacteria, preadsorbed on magnetite, to TiO(2) particles. The temporary co-adhesion to magnetite and TiO(2) was exploited for the design of a prototype biomagnetic separation process in which bacterial cells serve as an adhesive mediator between magnetite and TiO(2) particles in a mixture of Al, Si, and Ti oxides that simulates their proportion in the ash.

  20. Transcriptomic Assessment of Isozymes in the Biphenyl Pathway of Rhodococcus sp. Strain RHA1†

    PubMed Central

    Gonçalves, Edmilson R.; Hara, Hirofumi; Miyazawa, Daisuke; Davies, Julian E.; Eltis, Lindsay D.; Mohn, William W.

    2006-01-01

    Rhodococcus sp. RHA1 grows on a broad range of aromatic compounds and vigorously degrades polychlorinated biphenyls (PCBs). Previous work identified RHA1 genes encoding multiple isozymes for most of the seven steps of the biphenyl (BPH) pathway, provided evidence for coexpression of some of these isozymes, and indicated the involvement of some of these enzymes in the degradation of BPH, ethylbenzene (ETB), and PCBs. To investigate the expression of these isozymes and better understand how they contribute to the robust degradative capacity of RHA1, we comprehensively analyzed the 9.7-Mb genome of RHA1 for BPH pathway genes and characterized the transcriptome of RHA1 growing on benzoate (BEN), BPH, and ETB. Sequence analyses revealed 54 potential BPH pathway genes, including 28 not previously reported. Transcriptomic analysis with a DNA microarray containing 70-mer probes for 8,213 RHA1 genes revealed a suite of 320 genes of diverse functions that were upregulated during growth both on BPH and on ETB, relative to growth on the control substrate, pyruvate. By contrast, only 65 genes were upregulated during growth on BEN. Quantitative PCR assays confirmed microarray results for selected genes and indicated that some of the catabolic genes were upregulated over 10,000-fold. Our analysis suggests that up to 22 enzymes, including 8 newly identified ones, may function in the BPH pathway of RHA1. The relative expression levels of catabolic genes did not differ for BPH and ETB, suggesting a common regulatory mechanism. This study delineated a suite of catabolic enzymes for biphenyl and alkyl-benzenes in RHA1, which is larger than previously recognized and which may serve as a model for catabolism in other environmentally important bacteria having large genomes. PMID:16957245

  1. Methylated Cytokinins from the Phytopathogen Rhodococcus fascians Mimic Plant Hormone Activity.

    PubMed

    Radhika, Venkatesan; Ueda, Nanae; Tsuboi, Yuuri; Kojima, Mikiko; Kikuchi, Jun; Kudo, Takuji; Sakakibara, Hitoshi

    2015-10-01

    Cytokinins (CKs), a class of phytohormones that regulate plant growth and development, are also synthesized by some phytopathogens to disrupt the hormonal balance and to facilitate niche establishment in their hosts. Rhodococcus fascians harbors the fasciation (fas) locus, an operon encoding several genes homologous to CK biosynthesis and metabolism. This pathogen causes unique leafy gall symptoms reminiscent of CK overproduction; however, bacterial CKs have not been clearly correlated with the severe symptoms, and no virulence-associated unique CKs or analogs have been identified. Here, we report the identification of monomethylated N(6)-(∆(2)-isopentenyl)adenine and dimethylated N(6)-(∆(2)-isopentenyl)adenine (collectively, methylated cytokinins [MeCKs]) from R. fascians. MeCKs were recognized by a CK receptor and up-regulated type-A ARABIDOPSIS THALIANA RESPONSE REGULATOR genes. Treatment with MeCKs inhibited root growth, a hallmark of CK action, whereas the receptor mutant was insensitive. MeCKs were retained longer in planta than canonical CKs and were poor substrates for a CK oxidase/dehydrogenase, suggesting enhanced biological stability. MeCKs were synthesized by S-adenosyl methionine-dependent methyltransferases (MT1 and MT2) that are present upstream of the fas genes. The best substrate for methylation was isopentenyl diphosphate. MT1 and MT2 catalyzed distinct methylation reactions; only the MT2 product was used by FAS4 to synthesize monomethylated N(6)-(∆(2)-isopentenyl)adenine. The MT1 product was dimethylated by MT2 and used as a substrate by FAS4 to produce dimethylated N(6)-(∆(2)-isopentenyl)adenine. Chemically synthesized MeCKs were comparable in activity. Our results strongly suggest that MeCKs function as CK mimics and play a role in this plant-pathogen interaction.

  2. Methylated Cytokinins from the Phytopathogen Rhodococcus fascians Mimic Plant Hormone Activity1[OPEN

    PubMed Central

    Radhika, Venkatesan; Ueda, Nanae; Tsuboi, Yuuri; Kojima, Mikiko; Kikuchi, Jun; Kudo, Takuji; Sakakibara, Hitoshi

    2015-01-01

    Cytokinins (CKs), a class of phytohormones that regulate plant growth and development, are also synthesized by some phytopathogens to disrupt the hormonal balance and to facilitate niche establishment in their hosts. Rhodococcus fascians harbors the fasciation (fas) locus, an operon encoding several genes homologous to CK biosynthesis and metabolism. This pathogen causes unique leafy gall symptoms reminiscent of CK overproduction; however, bacterial CKs have not been clearly correlated with the severe symptoms, and no virulence-associated unique CKs or analogs have been identified. Here, we report the identification of monomethylated N6-(∆2-isopentenyl)adenine and dimethylated N6-(∆2-isopentenyl)adenine (collectively, methylated cytokinins [MeCKs]) from R. fascians. MeCKs were recognized by a CK receptor and up-regulated type-A ARABIDOPSIS THALIANA RESPONSE REGULATOR genes. Treatment with MeCKs inhibited root growth, a hallmark of CK action, whereas the receptor mutant was insensitive. MeCKs were retained longer in planta than canonical CKs and were poor substrates for a CK oxidase/dehydrogenase, suggesting enhanced biological stability. MeCKs were synthesized by S-adenosyl methionine-dependent methyltransferases (MT1 and MT2) that are present upstream of the fas genes. The best substrate for methylation was isopentenyl diphosphate. MT1 and MT2 catalyzed distinct methylation reactions; only the MT2 product was used by FAS4 to synthesize monomethylated N6-(∆2-isopentenyl)adenine. The MT1 product was dimethylated by MT2 and used as a substrate by FAS4 to produce dimethylated N6-(∆2-isopentenyl)adenine. Chemically synthesized MeCKs were comparable in activity. Our results strongly suggest that MeCKs function as CK mimics and play a role in this plant-pathogen interaction. PMID:26251309

  3. Purification, Characterization, and Overexpression of Flavin Reductase Involved in Dibenzothiophene Desulfurization by Rhodococcus erythropolis D-1

    PubMed Central

    Matsubara, Toshiyuki; Ohshiro, Takashi; Nishina, Yoshihiro; Izumi, Yoshikazu

    2001-01-01

    The dibenzothiophene (DBT)-desulfurizing bacterium, Rhodococcus erythropolis D-1, removes sulfur from DBT to form 2-hydroxybiphenyl using four enzymes, DszC, DszA, DszB, and flavin reductase. In this study, we purified and characterized the flavin reductase from R. erythropolis D-1 grown in a medium containing DBT as the sole source of sulfur. It is conceivable that the enzyme is essential for two monooxygenase (DszC and DszA) reactions in vivo. The purified flavin reductase contains no chromogenic cofactors and was found to have a molecular mass of 86 kDa and four identical 22-kDa subunits. The enzyme catalyzed NADH-dependent reduction of flavin mononucleotide (FMN), and the Km values for NADH and FMN were 208 and 10.8 μM, respectively. Flavin adenine dinucleotide was a poor substrate, and NADPH was inert. The enzyme did not catalyze reduction of any nitroaromatic compound. The optimal temperature and optimal pH for enzyme activity were 35°C and 6.0, respectively, and the enzyme retained 30% of its activity after heat treatment at 80°C for 30 min. The N-terminal amino acid sequence of the purified flavin reductase was identical to that of DszD of R. erythropolis IGTS8 (K. A. Gray, O. S. Pogrebinsky, G. T. Mrachko, L. Xi, D. J. Monticello, and C. H. Squires, Nat. Biotechnol. 14:1705–1709, 1996). The flavin reductase gene was amplified with primers designed by using dszD of R. erythropolis IGTS8, and the enzyme was overexpressed in Escherichia coli. The specific activity in crude extracts of the overexpressed strain was about 275-fold that of the wild-type strain. PMID:11229908

  4. γ-Resorcylate Catabolic-Pathway Genes in the Soil Actinomycete Rhodococcus jostii RHA1

    PubMed Central

    Kasai, Daisuke; Araki, Naoto; Motoi, Kota; Yoshikawa, Shota; Iino, Toju; Imai, Shunsuke; Masai, Eiji

    2015-01-01

    The Rhodococcus jostii RHA1 gene cluster required for γ-resorcylate (GRA) catabolism was characterized. The cluster includes tsdA, tsdB, tsdC, tsdD, tsdR, tsdT, and tsdX, which encode GRA decarboxylase, resorcinol 4-hydroxylase, hydroxyquinol 1,2-dioxygenase, maleylacetate reductase, an IclR-type regulator, a major facilitator superfamily transporter, and a putative hydrolase, respectively. The tsdA gene conferred GRA decarboxylase activity on Escherichia coli. Purified TsdB oxidized NADH in the presence of resorcinol, suggesting that tsdB encodes a unique NADH-specific single-component resorcinol 4-hydroxylase. Mutations in either tsdA or tsdB resulted in growth deficiency on GRA. The tsdC and tsdD genes conferred hydroxyquinol 1,2-dioxygenase and maleylacetate reductase activities, respectively, on E. coli. Inactivation of tsdT significantly retarded the growth of RHA1 on GRA. The growth retardation was partially suppressed under acidic conditions, suggesting the involvement of tsdT in GRA uptake. Reverse transcription-PCR analysis revealed that the tsd genes constitute three transcriptional units, the tsdBADC and tsdTX operons and tsdR. Transcription of the tsdBADC and tsdTX operons was induced during growth on GRA. Inactivation of tsdR derepressed transcription of the tsdBADC and tsdTX operons in the absence of GRA, suggesting that tsd gene transcription is negatively regulated by the tsdR-encoded regulator. Binding of TsdR to the tsdR-tsdB and tsdT-tsdR intergenic regions was inhibited by the addition of GRA, indicating that GRA interacts with TsdR as an effector molecule. PMID:26319878

  5. Foal Monocyte-Derived Dendritic Cells Become Activated upon Rhodococcus equi Infection▿ †

    PubMed Central

    Flaminio, M. Julia B. F.; Nydam, Daryl V.; Marquis, Hélène; Matychak, Mary Beth; Giguère, Steeve

    2009-01-01

    Susceptibility of foals to Rhodococcus equi pneumonia is exclusive to the first few months of life. The objective of this study was to investigate the immediate immunologic response of foal and adult horse antigen-presenting cells (APCs) upon infection with R. equi. We measured the activation of the antigen-presenting major histocompatibility complex (MHC) class II molecule, costimulatory molecules CD40 and CD86, the cytokine interleukin-12 (IL-12), and the transcriptional factor interferon regulatory factor 1 (IRF-1) in monocyte-derived macrophages (mMOs) and dendritic cells (mDCs) of adult horses and foals of different ages (from birth to 3 months of age) infected with virulent R. equi or its avirulent, plasmid-cured derivative. Infection with virulent or avirulent R. equi induced (P ≤ 0.01) the expression of IL-12p35 and IL-12p40 mRNAs in foal mMOs and mDCs at different ages. This response was likely mediated by the higher (P = 0.008) expression of IRF-1 in foal mDCs at birth than in adult horse mDCs. R. equi infection promoted comparable expression of costimulatory molecules CD86 and CD40 in foal and adult horse cells. The cytokine and costimulatory response by foal mDCs was not accompanied by robust MHC class II molecule expression. These data suggest that foal APCs detect the presence of R. equi and respond with the expression of the Th1-inducing cytokine IL-12. Nevertheless, there seems to be a limitation to MHC class II molecule expression which we hypothesize may compromise the efficient priming of naïve effector cells in early life. PMID:19109450

  6. Ethyl tert-butyl ether (ETBE) biodegradation by a syntrophic association of Rhodococcus sp. IFP 2042 and Bradyrhizobium sp. IFP 2049 isolated from a polluted aquifer.

    PubMed

    Le Digabel, Yoann; Demanèche, Sandrine; Benoit, Yves; Vogel, Timothy M; Fayolle-Guichard, Françoise

    2013-12-01

    Ethyl tert-butyl ether (ETBE) enrichment was obtained by adding contaminated groundwater to a mineral medium containing ETBE as the sole carbon and energy source. ETBE was completely degraded to biomass and CO2 with a transient production of tert-butanol (TBA) and a final biomass yield of 0.37 ± 0.08 mg biomass (dry weight).mg(-1) ETBE. Two bacterial strains, IFP 2042 and IFP 2049, were isolated from the enrichment, and their 16S rRNA genes (rrs) were similar to Rhodococcus sp. (99 % similarity to Rhodococcus erythropolis) and Bradyrhizobium sp. (99 % similarity to Bradyrhizobium japonicum), respectively. Rhodococcus sp. IFP 2042 degraded ETBE to TBA, and Bradyrhizobium sp. IFP 2049 degraded TBA to biomass and CO2. A mixed culture of IFP 2042 and IFP 2049 degraded ETBE to CO2 with a biomass yield similar to the original ETBE enrichment (0.31 ± 0.02 mg biomass.mg(-1) ETBE). Among the genes previously described to be involved in ETBE, MTBE, and TBA degradation, only alkB was detected in Rhodococcus sp. IFP 2042 by PCR, and none were detected in Bradyrhizobium sp. IFP 2049.

  7. A Case of Recurrent Meningitis Caused by Rhodococcus species Successfully Treated with Antibiotic Treatment and Intrathecal Injection of Vancomycin through an Ommaya Reservoir

    PubMed Central

    Lee, Kanglok; Rho, Min; Yu, Miyeon; Kwak, Joohee; Hong, Seungpyo; Kim, Jisoong; Kim, Yeonjae

    2015-01-01

    Human infection by Rhodococcus species is rare and mostly limited to immunocompromised hosts such as patients infected with the human immunodeficiency virus (HIV) or organ transplant recipients. The most common strain is R. equi, and the most common clinical presentation is pulmonary infection, reported in 80% of Rhodococcus spp. infections. The central nervous system is an uncommon infection site. We report a case of a patient with pneumonia, brain abscess, and recurrent meningitis caused by Rhodococcus spp. He initially presented with pneumonia with necrosis, which progressed to brain abscess and recurrent meningitis. Rhodococcus spp. was identified from the cerobrospinal fluid (CSF) collected during his fourth hospital admission. Despite prolonged treatment with appropriate antibiotics, meningitis recurred three times. Finally, in order to administer antibiotics directly into the CSF and bypass the blood-brain barrier, an Ommaya reservoir was inserted for administration of 90 days of intrathecal vancomycin and amikacin in conjunction with intravenous and oral antibiotics; the patient was finally cured with this treatment regimen. PMID:26483993

  8. Draft Genome Sequence of Rhodococcus erythropolis VSD3, a Diesel Fuel-Degrading and Plant Growth-Promoting Bacterium Isolated from Hedera helix Leaves.

    PubMed

    Stevens, Vincent; Thijs, Sofie; McAmmond, Breanne; Langill, Tori; Van Hamme, Jonathan; Weyens, Nele; Vangronsveld, Jaco

    2017-02-23

    We report here the 6.55-Mb draft genome sequence of Rhodococcus erythropolis VSD3, a Gram-positive bacterium of the Nocardiaceae family, isolated from leaves of Hedera helix growing at a high-traffic city center in Belgium. The exploration of its genome will contribute to the assessment of its application as an inoculant in phylloremediation approaches.

  9. Draft Genome Sequence of Rhodococcus rhodochrous Strain KG-21, a Soil Isolate from Oil Fields of Krishna-Godavari Basin, India.

    PubMed

    Dawar, Chhavi; Aggarwal, Ramesh K

    2015-10-15

    Here, we present the 6.1-Mb draft genome sequence of Rhodococcus rhodochrous strain KG-21, a soil isolate from the oil fields of Krishna-Godavari Basin in Andhra Pradesh, India. This genomic resource may help in the identification of the gene(s) involved in hydrocarbon degradation and their possible deployment for bioremediation.

  10. Degradation of 4-nitrophenol, 2-chloro-4-nitrophenol, and 2,4-dinitrophenol by Rhodococcus imtechensis strain RKJ300.

    PubMed

    Ghosh, Anuradha; Khurana, Meenu; Chauhan, Archana; Takeo, Masahiro; Chakraborti, Asit K; Jain, Rakesh K

    2010-02-01

    A bacterial strain Rhodococcus imtechensis RKJ300 (= MTCC 7085(T) = JCM 13270(T)) was isolated from pesticide-contaminated soil of Punjab by the enrichment technique on minimal medium containing 4-nitrophenol. Strain RKJ300 is capable of utilizing 4-nitrophenol, 2-chloro-4-nitrophenol, and 2,4-dinitrophenol as sole sources of carbon and energy. The strain involved both oxidative and reductive catabolic mechanisms for initial transformation of these compounds. In the case of 2-chloro-4-nitrophenol, colorimetric analysis indicated that nitrite release was followed by stoichiometric elimination of chloride ions. Experiments using whole cells and cell-free extracts showed chlorohydroquinone and hydroquinone as the intermediates of 2-chloro-4-nitrophenol degradation. This is the first report of degradation on 2-chloro-4-nitrophenol by a bacterium under aerobic condition to the best of our knowledge. However, pathways for degradation of 4-nitrophenol and 2,4-dinitrophenol were similar to those reported in other strains of Rhodococcus. Laboratory-scale soil microcosm studies demonstrated that the organism was capable of degrading a mixture of nitrophenols simultaneously, indicating its applicability toward in situ bioremediation of contaminated sites. The fate of the augmented strain as monitored by the plate-counting method and hybridization technique was found to be fairly stable throughout the period of microcosm experiments.

  11. Biotransformation of d-Limonene to (+) trans-Carveol by Toluene-Grown Rhodococcus opacus PWD4 Cells

    PubMed Central

    Duetz, Wouter A.; Fjällman, Ann H. M.; Ren, Shuyu; Jourdat, Catherine; Witholt, Bernard

    2001-01-01

    The toluene-degrading strain Rhodococcus opacus PWD4 was found to hydroxylate d-limonene exclusively in the 6-position, yielding enantiomerically pure (+) trans-carveol and traces of (+) carvone. This biotransformation was studied using cells cultivated in chemostat culture with toluene as a carbon and energy source. The maximal specific activity of (+) trans-carveol formation was 14.7 U (g of cells [dry weight])−1, and the final yield was 94 to 97%. Toluene was found to be a strong competitive inhibitor of the d-limonene conversion. Glucose-grown cells did not form any trans-carveol from d-limonene. These results suggest that one of the enzymes involved in toluene degradation is responsible for this allylic monohydroxylation. Another toluene degrader (Rhodococcus globerulus PWD8) had a lower specific activity but was found to oxidize most of the formed trans-carveol to (+) carvone, allowing for the biocatalytic production of this flavor compound. PMID:11375201

  12. Development of ELISA test for determination of the level of antibodies against Rhodococcus equi in equine serum and colostrum.

    PubMed

    Witkowski, Lucjan; Kaba, Jarosław; Rzewuska, Magdalena; Nowicki, Mariusz; Szaluś-Jordanow, Olga; Kita, Jerzy

    2012-10-15

    Rhodococcus equi infection occurs worldwide and is one of the major causes of losing foals in the first six months of life. The application of serological tests in the diagnostics of rhodococcosis is limited, however they play a crucial role in immunological studies. The objective of this study was to develop and standardize ELISA test for the determination of the level of antibodies against Rhodococcus equi in equine serum and colostrum.Bacterial cell lysate was used as antigen. The test was standardized on 175 sera obtained from adult horses kept on rhodococcosis-free and endemic farms. Positive and negative control sera were used. The test detected IgG antibodies mainly against VapA protein, which was confirmed by Western blot analysis. The test was easy to perform, did not require inactivation of sera and had low well-to-well variation. The shelf life of antigen-coated ELISA plates was 21 days.The test allowed to reveal significant increase of R. equi-specific antibodies in both serum and colostrum in response to the vaccination (p<0.001). Therefore it can be applied to the evaluation of efficacy of immunization. Moreover, no statistically significant difference in the baseline antibody level in adult horses from rhodococcosis-free and endemic farm was revealed (α=0.05). Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Improved identification of Gordonia, Rhodococcus and Tsukamurella species by 5'-end 16S rRNA gene sequencing.

    PubMed

    Wang, Tao; Kong, Fanrong; Chen, Sharon; Xiao, Meng; Sorrell, Tania; Wang, Xiaoyan; Wang, Shuo; Sintchenko, Vitali

    2011-01-01

    The identification of fastidious aerobic Actinomycetes such as Gordonia, Rhodococcus, and Tsukamurella has remained a challenge leading to clinically significant misclassifications. This study is intended to examine the feasibility of partial 5'-end 16S rRNA gene sequencing for the identification of Gordonia, Rhodococcus, and Tsukamurella, and defined potential reference sequences for species from each of these genera. The 16S rRNA gene sequence based identification algorithm for species identification was used and enhanced by aligning test sequences with reference sequences from the List of Prokaryotic Names with Standing in Nomenclature. Conventional PCR based 16S rRNA gene sequencing and the alignment of the isolate 16S rRNA gene sequence with reference sequences accurately identified 100% of clinical strains of aerobic Actinomycetes. While partial 16S rRNA gene sequences of reference type strains matched with the 16S rRNA gene sequences of 19 isolates in our data set, another 13 strains demonstrated a degree of polymorphism with a 1-4 bp difference in the regions of difference. 5'-end 606 bp 16S rRNA gene sequencing, coupled with the assignment of well defined reference sequences to clinically relevant species of bacteria, can be a useful strategy for improving the identification of clinically relevant aerobic Actinomycetes.

  14. Enhanced biodegradation of diesel oil by a newly identified Rhodococcus baikonurensis EN3 in the presence of mycolic acid.

    PubMed

    Lee, M; Kim, M K; Singleton, I; Goodfellow, M; Lee, S-T

    2006-02-01

    The aim of the present study was to isolate and characterize a bacterium, strain EN3, capable of using diesel oil as a major carbon and energy source, and to analyse the enhancement of diesel oil degradation by this organism using synthetic mycolic acid (2-hexyl-3-hydroxyldecanoic acid). An actinomycete with the ability to degrade diesel oil was isolated from oil contaminated soil and characterized. The strain had phenotypic properties consistent with its classification in the genus Rhodococcus showing a 16S rRNA gene similarity of 99.7% with Rhodococcus baikonurensis DSM 44587(T). The ability of the characterized strain to degrade diesel oil at various concentrations (1000, 5000, 10 000 and 20 000 mg l(-1)) was determined. The effect of synthetic mycolic acid on the biodegradation of diesel oil was investigated at the 20 000 mg l(-1) concentration; the surfactant was added to the flask cultures at three different concentrations (10, 50 and 100 mg l(-1)) and degradation followed over 7 days. Enhanced degradation was found at all three concentrations of the surfactant. In addition, the enhancement of diesel oil degradation by other surfactants was observed. The synthetic mycolic acid has potential for the remediation of petroleum-contaminated sites from both an economic and applied perspective as it can stimulate biodegradation at low concentrations. This study showed that the synthesized mycolic acid can be used for potential applications in the bioremediation industries, for example, in oil spill clean-up, diesel fuel remediation and biostimulation.

  15. Homologous npdGI genes in 2,4-dinitrophenol- and 4-nitrophenol-degrading Rhodococcus spp.

    PubMed

    Heiss, Gesche; Trachtmann, Natalie; Abe, Yoshikatsu; Takeo, Masahiro; Knackmuss, Hans-Joachim

    2003-05-01

    Rhodococcus (opacus) erythropolis HL PM-1 grows on 2,4,6-trinitrophenol or 2,4-dinitrophenol (2,4-DNP) as a sole nitrogen source. The NADPH-dependent F(420) reductase (NDFR; encoded by npdG) and the hydride transferase II (HTII; encoded by npdI) of the strain were previously shown to convert both nitrophenols to their respective hydride Meisenheimer complexes. In the present study, npdG and npdI were amplified from six 2,4-DNP degrading Rhodococcus spp. The genes showed sequence similarities of 86 to 99% to the respective npd genes of strain HL PM-1. Heterologous expression of the npdG and npdI genes showed that they were involved in 2,4-DNP degradation. Sequence analyses of both the NDFRs and the HTIIs revealed conserved domains which may be involved in binding of NADPH or F(420). Phylogenetic analyses of the NDFRs showed that they represent a new group in the family of F(420)-dependent NADPH reductases. Phylogenetic analyses of the HTIIs revealed that they form an additional group in the family of F(420)-dependent glucose-6-phosphate dehydrogenases and F(420)-dependent N(5),N(10)-methylenetetrahydromethanopterin reductases. Thus, the NDFRs and the HTIIs may each represent a novel group of F(420)-dependent enzymes involved in catabolism.

  16. Transcriptomics and Lipidomics of the Environmental Strain Rhodococcus ruber Point out Consumption Pathways and Potential Metabolic Bottlenecks for Polyethylene Degradation.

    PubMed

    Gravouil, Kévin; Ferru-Clément, Romain; Colas, Steven; Helye, Reynald; Kadri, Linette; Bourdeau, Ludivine; Moumen, Bouziane; Mercier, Anne; Ferreira, Thierry

    2017-04-12

    Polyethylene (PE), one of the most prominent synthetic polymers used worldwide, is very poorly biodegradable in the natural environment. Consequently, PE represents by itself more than half of all plastic wastes. PE biodegradation is achieved through the combination of abiotic and biotic processes. Several microorganisms have been shown to grow on the surface of PE materials, among which are the species of the Rhodococcus genus, suggesting a potent ability of these microorganisms to use, at least partly, PE as a potent carbon source. However, most of them, if not all, fail to induce a clear-cut degradation of PE samples, showing that bottlenecks to reach optimal biodegradation clearly exist. To identify the pathways involved in PE consumption, we used in the present study a combination of RNA-sequencing and lipidomic strategies. We show that short-term exposure to various forms of PE, displaying different molecular weight distributions and oxidation levels, lead to an increase in the expression of 158 genes in a Rhodococcus representative, R. ruber. Interestingly, one of the most up-regulated pathways is related to alkane degradation and β-oxidation of fatty acids. This approach also allowed us to identify metabolic limiting steps, which could be fruitfully targeted for optimized PE consumption by R. ruber.

  17. Rhodococcus erythropolis DCL14 contains a novel degradation pathway for limonene.

    PubMed

    van der Werf, M J; Swarts, H J; de Bont, J A

    1999-05-01

    Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample. This organism was identified as a strain of Rhodococcus erythropolis by chemotaxonomic and genetic studies. R. erythropolis DCL14 also assimilated the terpenes limonene-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (-)-menthol, while perillyl alcohol was not utilized as a carbon and energy source. Induction tests with cells grown on limonene revealed that the oxygen consumption rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of R. erythropolis DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1, 2-monooxygenase activity, a cofactor-independent limonene-1, 2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation studies showed that (1S,2S,4R)-limonene-1,2-diol, (1S, 4R)-1-hydroxy-2-oxolimonene, and (3R)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)-limonene degradation pathway. The opposite enantiomers [(1R,2R,4S)-limonene-1,2-diol, (1R, 4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxoheptanoate] were found in the (4S)-limonene degradation pathway, while accumulation of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed. These results show that R. erythropolis DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone. This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate

  18. Identification of Rhodococcus equi lipids recognized by host cytotoxic T lymphocytes

    PubMed Central

    Harris, Seth P.; Fujiwara, Nagatoshi; Mealey, Robert H.; Alperin, Debra C.; Naka, Takashi; Goda, Reina

    2010-01-01

    Immune adult horses have CD8+ cytotoxic T lymphocytes (CTLs) that recognize and lyse Rhodococcus equi-infected cells in an equine lymphocyte alloantigen (ELA)-A [classical major histocompatibility complex (MHC) class I]-unrestricted fashion. As protein antigens are MHC class I-restricted, the lack of restriction suggests that the bacterial antigens being recognized by the host are not proteins. The goals of this study were to test the hypothesis that these CTLs recognize unique R. equi cell-wall lipids related to mycobacterial lipids. Initial experiments showed that treatment of soluble R. equi antigen with broadly reactive proteases did not significantly diminish the ability of the antigen to stimulate R. equi-specific CTLs. R. equi-specific CTLs were also shown to lyse target cells (equine macrophages) pulsed with an R. equi lipid extract. Analysis of the R. equi lipid by TLC and MS (MALDI-TOF and ES) indicated that the extracted antigen consisted of three primary fractions: trehalose monomycolate (TMM), trehalose dimycolate (TDM) and cardiolipin (CL). ELA-A-mismatched cells pulsed with purified TMM and CL, but not the TDM fraction, were recognized and lysed by R. equi-specific CTLs. Because of their role in immune clearance and pathogenesis, transcription of the cytokines gamma interferon (IFN-γ) and interleukin-4 (IL-4) was also measured in response to R. equi lipids by using real-time PCR; elevated IFN-γ, but not IL-4, was associated with host clearance of the bacteria. The whole-cell R. equi lipid and all three R. equi lipid fractions resulted in marked increases in IFN-γ transcription, but no increase in IL-4 transcription. Together, these data support the hypothesis that immune recognition of unique lipids in the bacterial cell wall is an important component of the protective immune response to R. equi. The results also identify potential lipid antigens not previously shown to be recognized by CTLs in an important, naturally occurring actinomycete

  19. Rhodococcus erythropolis DCL14 Contains a Novel Degradation Pathway for Limonene

    PubMed Central

    van der Werf, Mariët J.; Swarts, Henk J.; de Bont, Jan A. M.

    1999-01-01

    Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample. This organism was identified as a strain of Rhodococcus erythropolis by chemotaxonomic and genetic studies. R. erythropolis DCL14 also assimilated the terpenes limonene-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (−)-menthol, while perillyl alcohol was not utilized as a carbon and energy source. Induction tests with cells grown on limonene revealed that the oxygen consumption rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of R. erythropolis DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1,2-monooxygenase activity, a cofactor-independent limonene-1,2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation studies showed that (1S,2S,4R)-limonene-1,2-diol, (1S,4R)-1-hydroxy-2-oxolimonene, and (3R)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)-limonene degradation pathway. The opposite enantiomers [(1R,2R,4S)-limonene-1,2-diol, (1R,4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxoheptanoate] were found in the (4S)-limonene degradation pathway, while accumulation of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed. These results show that R. erythropolis DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone. This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate. In

  20. Engineering xylose metabolism in triacylglycerol-producing Rhodococcus opacus for lignocellulosic fuel production

    PubMed Central

    2013-01-01

    Background There has been a great deal of interest in fuel productions from lignocellulosic biomass to minimize the conflict between food and fuel use. The bioconversion of xylose, which is the second most abundant sugar present after glucose in lignocellulosic biomass, is important for the development of cost effective bioprocesses to fuels. Rhodococcus opacus PD630, an oleaginous bacterium, accumulates large amounts of triacylglycerols (TAGs), which can be processed into advanced liquid fuels. However, R. opacus PD630 does not metabolize xylose. Results We generated DNA libraries from a Streptomyces bacterium capable of utilizing xylose and introduced them into R. opacus PD630. Xsp8, one of the engineered strains, was capable of growing on up to 180 g L-1 of xylose. Xsp8 grown in batch-cultures derived from unbleached kraft hardwood pulp hydrolysate containing 70 g L-1 total sugars was able to completely and simultaneously utilize xylose and glucose present in the lignocellulosic feedstock, and yielded 11.0 g L-1 of TAGs as fatty acids, corresponding to 45.8% of the cell dry weight. The yield of total fatty acids per gram of sugars consumed was 0.178 g, which consisted primarily of palmitic acid and oleic acid. The engineered strain Xsp8 was introduced with two heterologous genes from Streptomyces: xylA, encoding xylose isomerase, and xylB, encoding xylulokinase. We further demonstrated that in addition to the introduction and the concomitant expression of heterologous xylA and xylB genes, there is another molecular target in the R. opacus genome which fully enables the functionality of xylA and xylB genes to generate the robust xylose-fermenting strain capable of efficiently producing TAGs at high xylose concentrations. Conclusion We successfully engineered a R. opacus strain that is capable of completely utilizing high concentrations of xylose or mixed xylose/glucose simultaneously, and substantiated its suitability for TAG production. This study demonstrates

  1. Functional characterization of 3-ketosteroid 9α-hydroxylases in Rhodococcus ruber strain chol-4.

    PubMed

    Guevara, Govinda; Heras, Laura Fernández de Las; Perera, Julián; Llorens, Juana María Navarro

    2017-09-01

    The 3-Ketosteroid-9α-Hydroxylase, also known as KshAB [androsta-1,4-diene-3,17-dione, NADH:oxygen oxidoreductase (9α-hydroxylating); EC 1.14.13.142)], is a key enzyme in the general scheme of the bacterial steroid catabolism in combination with a 3-ketosteroid-Δ(1)-dehydrogenase activity (KstD), being both responsible of the steroid nucleus (rings A/B) breakage. KshAB initiates the opening of the steroid ring by the 9α-hydroxylation of the C9 carbon of 4-ene-3-oxosteroids (e.g. AD) or 1,4-diene-3-oxosteroids (e.g. ADD), transforming them into 9α-hydroxy-4-androsten-3,17-dione (9OHAD) or 9α-hydroxy-1,4-androstadiene-3,17-dione (9OHADD), respectively. The redundancy of these enzymes in the actinobacterial genomes results in a serious difficulty for metabolic engineering this catabolic pathway to obtain intermediates of industrial interest. In this work, we have identified three homologous kshA genes and one kshB gen in different genomic regions of R. ruber strain Chol-4. We present a set of data that helps to understand their specific roles in this strain, including: i) description of the KshAB enzymes ii) construction and characterization of ΔkshB and single, double and triple ΔkshA mutants in R. ruber iii) growth studies of the above strains on different substrates and iv) genetic complementation and biotransformation assays with those strains. Our results show that KshA2 isoform is needed for the degradation of steroid substrates with short side chain, while KshA3 works on those molecules with longer side chains. KshA1 is a more versatile enzyme related to the cholic acid catabolism, although it also collaborates with KshA2 or KshA3 activities in the catabolism of steroids. Accordingly to what it is described for other Rhodococcus strains, our results also suggest that the side chain degradation is KshAB-independent. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Functional differentiation of 3-ketosteroid Δ(1)-dehydrogenase isozymes in Rhodococcus ruber strain Chol-4.

    PubMed

    Guevara, Govinda; Fernández de Las Heras, Laura; Perera, Julián; Navarro Llorens, Juana María

    2017-03-14

    The Rhodococcus ruber strain Chol-4 genome contains at least three putative 3-ketosteroid Δ(1)-dehydrogenase ORFs (kstD1, kstD2 and kstD3) that code for flavoenzymes involved in the steroid ring degradation. The aim of this work is the functional characterization of these enzymes prior to the developing of different biotechnological applications. The three R. ruber KstD enzymes have different substrate profiles. KstD1 shows preference for 9OHAD and testosterone, followed by progesterone, deoxy corticosterone AD and, finally, 4-BNC, corticosterone and 19OHAD. KstD2 shows maximum preference for progesterone followed by 5α-Tes, DOC, AD testosterone, 4-BNC and lastly 19OHAD, corticosterone and 9OHAD. KstD3 preference is for saturated steroid substrates (5α-Tes) followed by progesterone and DOC. A preliminary attempt to model the catalytic pocket of the KstD proteins revealed some structural differences probably related to their catalytic differences. The expression of kstD genes has been studied by RT-PCR and RT-qPCR. All the kstD genes are transcribed under all the conditions assayed, although an additional induction in cholesterol and AD could be observed for kstD1 and in cholesterol for kstD3. Co-transcription of some correlative genes could be stated. The transcription initiation signals have been searched, both in silico and in vivo. Putative promoters in the intergenic regions upstream the kstD1, kstD2 and kstD3 genes were identified and probed in an apramycin-promoter-test vector, leading to the functional evidence of those R. ruber kstD promoters. At least three putative 3-ketosteroid Δ(1)-dehydrogenase ORFs (kstD1, kstD2 and kstD3) have been identified and functionally confirmed in R. ruber strain Chol-4. KstD1 and KstD2 display a wide range of substrate preferences regarding to well-known intermediaries of the cholesterol degradation pathway (9OHAD and AD) and other steroid compounds. KstD3 shows a narrower substrate range with a preference for saturated

  3. Improved glycerol utilization by a triacylglycerol-producing Rhodococcus opacus strain for renewable fuels.

    PubMed

    Kurosawa, Kazuhiko; Radek, Andreas; Plassmeier, Jens K; Sinskey, Anthony J

    2015-01-01

    Glycerol generated during renewable fuel production processes is potentially an attractive substrate for the production of value-added materials by fermentation. An engineered strain MITXM-61 of the oleaginous bacterium Rhodococcus opacus produces large amounts of intracellular triacylglycerols (TAGs) for lipid-based biofuels on high concentrations of glucose and xylose. However, on glycerol medium, MITXM-61 does not produce TAGs and grows poorly. The aim of the present work was to construct a TAG-producing R. opacus strain capable of high-cell-density cultivation at high glycerol concentrations. An adaptive evolution strategy was applied to improve the conversion of glycerol to TAGs in R. opacus MITXM-61. An evolved strain, MITGM-173, grown on a defined medium with 16 g L(-1) glycerol, produced 2.3 g L(-1) of TAGs, corresponding to 40.4% of the cell dry weight (CDW) and 0.144 g g(-1) of TAG yield per glycerol consumed. MITGM-173 was able to grow on high concentrations (greater than 150 g L(-1)) of glycerol. Cultivated in a medium containing an initial concentration of 20 g L(-1) glycerol, 40 g L(-1) glucose, and 40 g L(-1) xylose, MITGM-173 was capable of simultaneously consuming the mixed substrates and yielding 13.6 g L(-1) of TAGs, representing 51.2% of the CDM. In addition, when 20 g L(-1) glycerol was pulse-loaded into the culture with 40 g L(-1) glucose and 40 g L(-1) xylose at the stationary growth phase, MITGM-173 produced 14.3 g L(-1) of TAGs corresponding to 51.1% of the CDW although residual glycerol in the culture was observed. The addition of 20 g L(-1) glycerol in the glucose/xylose mix resulted in a TAG yield per glycerol consumed of 0.170 g g(-1) on the initial addition and 0.279 g g(-1) on the pulse addition of glycerol. We have generated a TAG-producing R. opacus MITGM-173 strain that shows significantly improved glycerol utilization in comparison to the parental strain. The present study demonstrates that the

  4. Formation of intracytoplasmic lipid inclusions by Rhodococcus opacus strain PD630.

    PubMed

    Alvarez, H M; Mayer, F; Fabritius, D; Steinbüchel, A

    1996-06-01

    An oleaginous hydrocarbon-degrading Rhodococcus opacus strain (PD630) was isolated from a soil sample. The cells were able to grow on a variety of substrates and to produce large amounts of three different types of intracellular inclusions during growth on alkanes, phenylalkanes, or non-hydrocarbon substrates. Electron microscopy revealed large numbers of electron-transparent inclusions with a sphere-like structure. In addition, electron-dense inclusions representing polyphosphate and electron-transparent inclusions with an elongated disc-shaped morphology occurred in small amounts. The electron-transparent inclusions of alkane- or gluconate-grown cells were composed of neutral lipids (98%, w/w), phospholipids (1.2%, w/w), and protein (0.8%, w/w). The major component of the cellular inclusions was triacylglycerols; minor amounts of diacylglycerols and probably also some free fatty acids were also present. Free fatty acids and/or fatty acids in acylglycerols in cells of R. opacus amounted up to 76 or 87% of the cellular dry weight in gluconate- or olive-oil-grown cells, respectively. The fatty acid composition of the inclusions depended on the substrate used for cultivation. In cells cultivated on n-alkanes, the composition of the fatty acids was related to the substrate, and intermediates of the beta-oxidation pathway, such as hexadecanoic or pentadecanoic acid, were among the acylglycerols. Hexadecanoic acid was also the major fatty acid (up 36% of total fatty acids) occurring in the lipid inclusions of gluconate-grown cells. This indicated that strain PD630 utilized beta-oxidation and de novo fatty acid biosynthesis for the synthesis of storage lipids. Inclusions isolated from phenyldecane-grown cells contained mainly the non-modified substrate and phenylalkanoic acids derived from the hydrocarbon oxidation, such as phenyldecanoic acid, phenyloctanoic acid, and phenylhexanoic acid, and approximately 5% (w/w) of diacylglycerols. The lipid inclusions seemed to have

  5. Tolerance and adaptive evolution of triacylglycerol-producing Rhodococcus opacus to lignocellulose-derived inhibitors.

    PubMed

    Kurosawa, Kazuhiko; Laser, Josephine; Sinskey, Anthony J

    2015-01-01

    Lignocellulosic biomass has been investigated as a renewable non-food source for production of biofuels. A significant technical challenge to using lignocellulose is the presence of microbial growth inhibitors generated during pretreatment processes. Triacylglycerols (TAGs) are potential precursors for lipid-based biofuel production. Rhodococcus opacus MITXM-61 is an oleaginous bacterium capable of producing large amounts of TAGs on high concentrations of glucose and xylose present in lignocellulosic hydrolysates. However, this strain is sensitive to ligonocellulose-derived inhibitors. To understand the toxic effects of the inhibitors in lignocellulosic hydrolysates, strain MITXM-61 was examined for tolerance toward the potential inhibitors and was subjected to adaptive evolution for the resistance to the inhibitors. We investigated growth-inhibitory effects by potential lignocellulose-derived inhibitors of phenols (lignin, vanillin, 4-hydroxybenzaldehyde (4-HB), syringaldehyde), furans (furfural and 5-hydroxymethyl-2-furaldehyde), and organic acids (levulinic acid, formic acid, and acetic acid) on the growth and TAG production of strain MITXM-61. Phenols and furans exhibited potent inhibitory effects at a concentration of 1 g L(-1), while organic acids had insignificant impacts at concentrations of up to 2 g L(-1). In an attempt to improve the inhibitor tolerance of strain MITXM-61, we evaluated the adaptation of this strain to the potential inhibitors. Adapted mutants were generated on defined agar media containing lignin, 4-HB, and syringaldehyde. Strain MITXM-61(SHL33) with improved multiple resistance of lignin, 4-HB, and syringaldehyde was constructed through adaptive evolution-based strategies. The evolved strain exhibited a two- to threefold increase in resistance to lignin, 4-HB, and syringaldehyde at 50% growth-inhibitory concentrations, compared to the parental strain. When grown in genuine lignocellulosic hydrolysates of corn stover, wheat straw, and

  6. Comparative genomic analyses reveal a lack of a substantial signature of host adaptation in Rhodococcus equi ('Prescottella equi').

    PubMed

    Sangal, Vartul; Jones, Amanda L; Goodfellow, Michael; Sutcliffe, Iain C; Hoskisson, Paul A

    2014-08-01

    Rhodococcus equi ('Prescottella equi') is a pathogenic actinomycete primarily infecting horses but has emerged as an opportunistic human pathogen. We have sequenced the genome of the type strain of this species, R. equi strain C7(T) , and compared the genome with that of another foal isolate 103S and of a human isolate ATCC 33707. The R. equi strains are closely related to each other and yet distantly related to other rhodococci and Nocardia brasiliensis. The comparison of gene contents among R. equi strains revealed minor differences that could be associated with host adaptation from foals to humans, including the presence of a paa operon in the human isolate, which is potentially involved in pathogenesis. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  7. Identification of Virulence-Associated Plasmids in Rhodococcus equi in Humans with and without Acquired Immunodeficiency Syndrome in Brazil

    PubMed Central

    Ribeiro, Márcio Garcia; Takai, Shinji; de Vargas, Agueda Castagna; Mattos-Guaraldi, Ana Luiza; Ferreira Camello, Thereza Cristina; Ohno, Ryoko; Okano, Hajime; da Silva, Aristeu Vieira

    2011-01-01

    Virulence of Rhodococcus equi strains from 20 humans in Brazil was investigated by using a polymerase chain reaction to characterize isolates as virulent (VapA), intermediately virulent (VapB), and avirulent. Nine isolates were obtained from human immunodeficiency virus (HIV)–positive patients, six from HIV-negative patients, and five from patients of unknown status. Five isolates were VapB positive, four were VapA positive, and eleven were avirulent. Among the nine isolates from HIV-positive patients, five contained VapB plasmids and two contained VapA plasmids. Five VapB-positive isolates had the type 8 virulence plasmid. Eleven of the patients had a history of contact with livestock and/or a farm environment, and none had contact with pigs. PMID:21896813

  8. Safety and immunogenicity of a live-attenuated auxotrophic candidate vaccine against the intracellular pathogen Rhodococcus equi.

    PubMed

    Lopez, A M; Townsend, H G G; Allen, A L; Hondalus, M K

    2008-02-13

    Rhodococcus equi causes serious pneumonia in neonatal foals and is an opportunistic pathogen of people with compromised cellular immunity. No effective vaccine against R. equi disease in foals is available. We tested the safety and immunogenicity of a live, fully attenuated riboflavin auxotrophic candidate vaccine strain of R. equi (R. equi rib-). We demonstrated that R. equi rib- is immunogenic and capable of inducing IFN-gamma responses in immunocompetent BALB/c mice, yet it is safe even in an immunocompromised SCID mouse infection model. Moreover, it protects immunocompetent mice against virulent R. equi challenge. In foals, R. equi rib- was likewise safe and stimulated serum R. equi-specific immune responses. A preliminary immunization strategy did not afford protection against virulent R. equi challenge and therefore, optimization of the vaccine formulation and or vaccination protocol will be necessary.

  9. Biosynthesis of terephthalic acid, isophthalic acid and their derivatives from the corresponding dinitriles by tetrachloroterephthalonitrile-induced Rhodococcus sp.

    PubMed

    He, Yu-Cai; Wu, Ya-Dong; Pan, Xue-He; Ma, Cui-Luan

    2014-02-01

    The nitrilase from Rhodococcus sp. CCZU10-1 catalyses the hydrolysis of dinitriles to acids without the formation of amides and cyanocarboxylic acids. It was induced by benzonitrile and its analogues (tetrachloroterephthalonitrile > ε-caprolactam > benzonitrile > phenylacetonitrile), and had activity towards aromatic nitriles (terephthalonitrile > tetrachloroterephthalonitrile > isophthalonitrile > tetrachloroisophthalonitrile > tetrafluoroterephthalonitrile > benzonitrile). After the optimization, the highest nitrilase induction [311 U/(g DCW)] was achieved with tetrachloroterephthalonitrile (1 mM) in the medium after 24 h at 30 °C after optimum enzyme activity was at pH 6.8 and at 30 °C. Efficient biocatalyst recycling was achieved by cell immobilization in calcium alginate, with a product-to-biocatalyst ratios of 776 g terephthalic acid/g DCW and 630 g isophthalic acid/g DCW.

  10. Immobilization of Rhodococcus rhodochrous BX2 (an acetonitrile-degrading bacterium) with biofilm-forming bacteria for wastewater treatment.

    PubMed

    Li, Chunyan; Li, Yue; Cheng, Xiaosong; Feng, Liping; Xi, Chuanwu; Zhang, Ying

    2013-03-01

    In this study, a unique biofilm consisting of three bacterial strains with high biofilm-forming capability (Bacillus subtilis E2, E3, and N4) and an acetonitrile-degrading bacterium (Rhodococcus rhodochrous BX2) was established for acetonitrile-containing wastewater treatment. The results indicated that this biofilm exhibited strong resistance to acetonitrile loading shock and displayed a typical spatial and structural heterogeneity and completely depleted the initial concentration of acetonitrile (800mgL(-1)) within 24h in a moving-bed-biofilm reactor (MBBR) after operation for 30days. The immobilization of BX2 cells in the biofilm was confirmed by PCR-DGGE. It has been demonstrated that biofilm-forming bacteria can promote the immobilization of contaminant-degrading bacteria in the biofilms and can subsequently improve the degradation of contaminants in wastewater. This approach offers a novel strategy for enhancing biological oxidation of toxic pollutants in wastewater.

  11. Identification of Atypical Rhodococcus-Like Clinical Isolates as Dietzia spp. by 16S rRNA Gene Sequencing▿

    PubMed Central

    Pilares, Lilian; Agüero, Jesús; Vázquez-Boland, José A.; Martínez-Martínez, Luis; Navas, Jesús

    2010-01-01

    Rhodococcus equi and Dietzia spp. are closely related actinomycetes that show similar phenotypic properties. In humans, R. equi is an opportunistic pathogen associated with severe immunodeficiency. Dietzia spp. are environmental bacteria that have been isolated recently from clinical material and are presumptively associated with human infections. During the last 5 years, 15 bacterial isolates from human clinical samples collected at the Hospital Marqués de Valdecilla, Santander, Spain, were identified as R. equi by the API Coryne test. 16S rRNA gene sequencing confirmed seven isolates to be true R. equi strains, whereas the other eight were identified as members of the genus Dietzia, including Dietzia maris (four isolates), Dietzia natronolimnaea (two isolates), and Dietzia timorensis and Dietzia sp. (one isolate each). The eight Dietzia isolates were highly sensitive to 12 antimicrobial compounds. PMID:20220156

  12. Metabolic shift in the phytopathogen Rhodococcus fascians in response to cell-free extract of infected tobacco plant tissues.

    PubMed

    Forizs, Laetitia; Lestrade, Sylvain; Mol, Adeline; Dierick, Jean-François; Gerbaux, Cécile; Diallo, Billo; El Jaziri, Mondher; Baucher, Marie; Vandeputte, Olivier M

    2009-05-01

    The phytopathogen Rhodococcus fascians induces the development of leafy gall, which is considered to be its ecological niche. To obtain a view of the metabolic changes occurring in R. fascians during this process, an in vitro system was used where bacteria are grown in the presence of a leafy gall extract, a condition mimicking that found by the bacteria in infected plants. Proteins of R. fascians grown for 24 h under these conditions were displayed by two-dimensional polyacrylamide gel electrophoresis. Fifteen polypeptides showing a differential accumulation in response to the inducing conditions were analyzed by mass spectrometry. Two polypeptides potentially linked to the Krebs cycle, a pyruvate dehydrogenase and a fumarate hydratase, were further characterized and shown to be downregulated at the transcriptional level. The identification of these two enzymes suggests that R. fascians may shift its metabolism during the interaction with plants from the Krebs cycle to the glyoxylate shunt.

  13. Virulence genes of the phytopathogen Rhodococcus fascians show specific spatial and temporal expression patterns during plant infection.

    PubMed

    Cornelis, Karen; Maes, Tania; Jaziri, Mondher; Holsters, Marcelle; Goethals, Koen

    2002-04-01

    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.

  14. Illegitimate integration of non-replicative vectors in the genome of Rhodococcus fascians upon electrotransformation as an insertional mutagenesis system.

    PubMed

    Desomer, J; Crespi, M; Van Montagu, M

    1991-09-01

    Electrotransformation of Rhodococcus fascians by non-replicating plasmids containing a suitable resistance marker resulted in stable transformants by integration of these constructs at various sites in the genome, thereby generating different mutations. Tagged genes could be isolated in Escherichia coli owing to the presence of a CoIE1 replicon and an ampicillin resistance gene in the inserted sequences. Southern analysis and nucleotide sequencing revealed that recombination can occur at defined locations in the plasmid, while no site preference for target sequences could be detected. Low homology between the recombining sequences indicates illegitimate recombination. The specificity of the plasmid sites could be explained by assuming a linear recombination intermediate, generated by cleavage of the transformed plasmid.

  15. Complete Nucleotide Sequence and Genetic Organization of the 210-Kilobase Linear Plasmid of Rhodococcus erythropolis BD2

    PubMed Central

    Stecker, Christiane; Johann, Andre; Herzberg, Christina; Averhoff, Beate; Gottschalk, Gerhard

    2003-01-01

    The complete nucleotide sequence of the linear plasmid pBD2 from Rhodococcus erythropolis BD2 comprises 210,205 bp. Sequence analyses of pBD2 revealed 212 putative open reading frames (ORFs), 97 of which had an annotatable function. These ORFs could be assigned to six functional groups: plasmid replication and maintenance, transport and metalloresistance, catabolism, transposition, regulation, and protein modification. Many of the transposon-related sequences were found to flank the isopropylbenzene pathway genes. This finding together with the significant sequence similarities of the ipb genes to genes of the linear plasmid-encoded biphenyl pathway in other rhodococci suggests that the ipb genes were acquired via transposition events and subsequently distributed among the rhodococci via horizontal transfer. PMID:12923100

  16. Pivalic acid acts as a starter unit in a fatty acid and antibiotic biosynthetic pathway in Alicyclobacillus, Rhodococcus and Streptomyces.

    PubMed

    Rezanka, Tomáš; Siristova, Lucie; Schreiberová, Olga; Rezanka, Michal; Masák, Jan; Melzoch, Karel; Sigler, Karel

    2011-06-01

    A biosynthetic pathway using pivalic acid as a starter unit was found in three bacterial species, Alicyclobacillus acidoterrestris, Rhodococcus erythropolis and Streptomyces avermitilis. When deuterium-labelled pivalic acid was added to A. acidoterrestris and R. erythropolis nutrient media it was incorporated into fatty acids to give rise to tert-butyl fatty acids (t-FAs). In addition, in R. erythropolis, pivalic acid was transformed into two starter units, i.e. isobutyric and 2-methylbutyric acid, which served as precursors of corresponding iso-even FAs and anteiso-FAs. In S. avermitilis the biosynthesis also yielded all three branched FAs; apart from this pathway, both pivalic and 2-methylbutyric acids were incorporated into the antibiotic avermectin. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

  17. Multiple reuses of Rhodococcus ruber TH3 free cells to produce acrylamide in a membrane dispersion microreactor.

    PubMed

    Li, Jiahui; Liu, Junqi; Chen, Jie; Wang, Yujun; Luo, Guangsheng; Yu, Huimin

    2015-01-01

    In this work, multiple reuses of Rhodococcus ruber TH3 free cells for the hydration of acrylonitrile to produce acrylamide in a membrane dispersion microreactor were carried out. Through using a centrifuge, the reactions reached 39.9, 39.5, 38.6 and 38.0wt% of the final acrylamide product concentration respectively within 35min in a four cycle reuse of free cells. In contrast, using a stirring tank, free cells could only be used once with the same addition speed of acrylonitrile with a microreactor. Through observing the dissolution behavior of acrylonitrile microdroplets in a free cell solution using a coaxial microfluidic device and microscope, it was found that the acrylonitrile microdroplets with a diameter of 75μm were rarely observed within a length of 2cm channel within 10s, which illustrated that the microreactor can intensify the reaction rate to reduce the inhibition of acrylonitrile and acrylamide.

  18. Effect of hyperimmune plasma on the severity of pneumonia caused by Rhodococcus equi in experimentally infected foals.

    PubMed

    Caston, Stephanie S; McClure, Scott R; Martens, Ronald J; Chaffin, M Keith; Miles, Kristina G; Griffith, Ronald W; Cohen, Noah D

    2006-01-01

    This study evaluated the prophylactic effectiveness of hyperimmune plasma (HIP) as an aid in the prevention of pneumonia caused by experimental infection with Rhodococcus equi. Thirty neonatal foals were administered R. equi HIP or saline at 2 days of age and were infected with virulent R. equi at 7 days. All foals developed signs or symptoms of respiratory disease. Radiographic scores on day 28 and neutrophil concentrations on day 49 were significantly greater in control foals, and time to respiratory effort score of 2 or higher was significantly shorter for control foals. Three foals, all in the principal group, died or were euthanized before the end of the study, but there was no significant difference in mortality between groups. VapA titers were significantly greater in principal foals. Administration of R. equi HIP decreased the severity of radiographic lesions and prolonged time to increased respiratory effort due to R. equi-induced pneumonia.

  19. An Invertron-Like Linear Plasmid Mediates Intracellular Survival and Virulence in Bovine Isolates of Rhodococcus equi

    PubMed Central

    Valero-Rello, Ana; Hapeshi, Alexia; Anastasi, Elisa; Alvarez, Sonsiray; Scortti, Mariela; Meijer, Wim G.; MacArthur, Iain

    2015-01-01

    We report a novel host-associated virulence plasmid in Rhodococcus equi, pVAPN, carried by bovine isolates of this facultative intracellular pathogenic actinomycete. Surprisingly, pVAPN is a 120-kb invertron-like linear replicon unrelated to the circular virulence plasmids associated with equine (pVAPA) and porcine (pVAPB variant) R. equi isolates. pVAPN is similar to the linear plasmid pNSL1 from Rhodococcus sp. NS1 and harbors six new vap multigene family members (vapN to vapS) in a vap pathogenicity locus presumably acquired via en bloc mobilization from a direct predecessor of equine pVAPA. Loss of pVAPN rendered R. equi avirulent in macrophages and mice. Mating experiments using an in vivo transconjugant selection strategy demonstrated that pVAPN transfer is sufficient to confer virulence to a plasmid-cured R. equi recipient. Phylogenetic analyses assigned the vap multigene family complement from pVAPN, pVAPA, and pVAPB to seven monophyletic clades, each containing plasmid type-specific allelic variants of a precursor vap gene carried by the nearest vap island ancestor. Deletion of vapN, the predicted “bovine-type” allelic counterpart of vapA, essential for virulence in pVAPA, abrogated pVAPN-mediated intramacrophage proliferation and virulence in mice. Our findings support a model in which R. equi virulence is conferred by host-adapted plasmids. Their central role is mediating intracellular proliferation in macrophages, promoted by a key vap determinant present in the common ancestor of the plasmid-specific vap islands, with host tropism as a secondary trait selected during coevolution with specific animal species. PMID:25895973

  20. An Invertron-Like Linear Plasmid Mediates Intracellular Survival and Virulence in Bovine Isolates of Rhodococcus equi.

    PubMed

    Valero-Rello, Ana; Hapeshi, Alexia; Anastasi, Elisa; Alvarez, Sonsiray; Scortti, Mariela; Meijer, Wim G; MacArthur, Iain; Vázquez-Boland, José A

    2015-07-01

    We report a novel host-associated virulence plasmid in Rhodococcus equi, pVAPN, carried by bovine isolates of this facultative intracellular pathogenic actinomycete. Surprisingly, pVAPN is a 120-kb invertron-like linear replicon unrelated to the circular virulence plasmids associated with equine (pVAPA) and porcine (pVAPB variant) R. equi isolates. pVAPN is similar to the linear plasmid pNSL1 from Rhodococcus sp. NS1 and harbors six new vap multigene family members (vapN to vapS) in a vap pathogenicity locus presumably acquired via en bloc mobilization from a direct predecessor of equine pVAPA. Loss of pVAPN rendered R. equi avirulent in macrophages and mice. Mating experiments using an in vivo transconjugant selection strategy demonstrated that pVAPN transfer is sufficient to confer virulence to a plasmid-cured R. equi recipient. Phylogenetic analyses assigned the vap multigene family complement from pVAPN, pVAPA, and pVAPB to seven monophyletic clades, each containing plasmid type-specific allelic variants of a precursor vap gene carried by the nearest vap island ancestor. Deletion of vapN, the predicted "bovine-type" allelic counterpart of vapA, essential for virulence in pVAPA, abrogated pVAPN-mediated intramacrophage proliferation and virulence in mice. Our findings support a model in which R. equi virulence is conferred by host-adapted plasmids. Their central role is mediating intracellular proliferation in macrophages, promoted by a key vap determinant present in the common ancestor of the plasmid-specific vap islands, with host tropism as a secondary trait selected during coevolution with specific animal species. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  1. Comparative Genomics of Rhodococcus equi Virulence Plasmids Indicates Host-Driven Evolution of the vap Pathogenicity Island.

    PubMed

    MacArthur, Iain; Anastasi, Elisa; Alvarez, Sonsiray; Scortti, Mariela; Vázquez-Boland, José A

    2017-05-01

    The conjugative virulence plasmid is a key component of the Rhodococcus equi accessory genome essential for pathogenesis. Three host-associated virulence plasmid types have been identified the equine pVAPA and porcine pVAPB circular variants, and the linear pVAPN found in bovine (ruminant) isolates. We recently characterized the R. equi pangenome (Anastasi E, et al. 2016. Pangenome and phylogenomic analysis of the pathogenic actinobacterium Rhodococcus equi. Genome Biol Evol. 8:3140-3148.) and we report here the comparative analysis of the virulence plasmid genomes. Plasmids within each host-associated type were highly similar despite their diverse origins. Variation was accounted for by scattered single nucleotide polymorphisms and short nucleotide indels, while larger indels-mostly in the plasticity region near the vap pathogencity island (PAI)-defined plasmid genomic subtypes. Only one of the plasmids analyzed, of pVAPN type, was exceptionally divergent due to accumulation of indels in the housekeeping backbone. Each host-associated plasmid type carried a unique PAI differing in vap gene complement, suggesting animal host-specific evolution of the vap multigene family. Complete conservation of the vap PAI was observed within each host-associated plasmid type. Both diversity of host-associated plasmid types and clonality of specific chromosomal-plasmid genomic type combinations were observed within the same R. equi phylogenomic subclade. Our data indicate that the overall strong conservation of the R. equi host-associated virulence plasmids is the combined result of host-driven selection, lateral transfer between strains, and geographical spread due to international livestock exchanges. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  2. Deep-sea Rhodococcus sp. BS-15, lacking the phytopathogenic fas genes, produces a novel glucotriose lipid biosurfactant.

    PubMed

    Konishi, Masaaki; Nishi, Shinro; Fukuoka, Tokuma; Kitamoto, Dai; Watsuji, Tomo-O; Nagano, Yuriko; Yabuki, Akinori; Nakagawa, Satoshi; Hatada, Yuji; Horiuchi, Jun-Ichi

    2014-08-01

    Glycolipid biosurfactant-producing bacteria were isolated from deep-sea sediment collected from the Okinawa Trough. Isolate BS15 produced the largest amount of the glycolipid, generating up to 6.31 ± 1.15 g l(-1) after 4 days at 20 °C. Glucose was identified in the hydrolysate of the purified major component of the biosurfactant glycolipid. According to gas chromatography/mass spectrometry analysis, the hydrophobic moieties in the major component were hexadecanoate, octadecanoate, 3-hydroxyhexadecanoate, 2-hydroxyoctanoate, and succinate. The molecular weight of the purified major glycolipid was calculated to be 1,211, while (1)H and (13)C nuclear magnetic resonance spectra confirmed that the major component consisted of 2 mol of α-glucoside and 1 mol of β-glucoside. The molecular structure was assigned as novel trisaccharide-type glycolipid biosurfactant, glucotriose lipids. The critical micelle concentration of the purified major glycolipid was 2.3 × 10(-6) M, with a surface tension of 29.5 mN m(-1). Phylogenetic analysis showed isolate BS15 was closely related to a Rhodococcus strains isolated from Antarctica, and to Rhodococcus fascians, a phytopathogen. PCR analysis showed that the fasA, fasB, fasC, fasD, fasE, and fasF genes, which are involved in phytohormone-like cytokinin production, were not present in the genome of BS15; however, analysis of a draft genome sequence of BS15 (5.5 Mb) identified regions with 31 %, 53 %, 46 %, 30 %, and 31 % DNA sequence identity to the fasA, fasB, fasC, and fasD genes, respectively.

  3. StyA1 and StyA2B from Rhodococcus opacus 1CP: a Multifunctional Styrene Monooxygenase System▿

    PubMed Central

    Tischler, Dirk; Kermer, René; Gröning, Janosch A. D.; Kaschabek, Stefan R.; van Berkel, Willem J. H.; Schlömann, Michael

    2010-01-01

    Two-component flavoprotein monooxygenases are emerging biocatalysts that generally consist of a monooxygenase and a reductase component. Here we show that Rhodococcus opacus 1CP encodes a multifunctional enantioselective flavoprotein monooxygenase system composed of a single styrene monooxygenase (SMO) (StyA1) and another styrene monooxygenase fused to an NADH-flavin oxidoreductase (StyA2B). StyA1 and StyA2B convert styrene and chemical analogues to the corresponding epoxides at the expense of FADH2 provided from StyA2B. The StyA1/StyA2B system presents the highest monooxygenase activity in an equimolar ratio of StyA1 and StyA2B, indicating (transient) protein complex formation. StyA1 is also active when FADH2 is supplied by StyB from Pseudomonas sp. VLB120 or PheA2 from Rhodococcus opacus 1CP. However, in both cases the reductase produces an excess of FADH2, resulting in a high waste of NADH. The epoxidation rate of StyA1 heavily depends on the type of reductase. This supports that the FADH2-induced activation of StyA1 requires interprotein communication. We conclude that the StyA1/StyA2B system represents a novel type of multifunctional flavoprotein monooxygenase. Its unique mechanism of cofactor utilization provides new opportunities for biotechnological applications and is highly relevant from a structural and evolutionary point of view. PMID:20675468

  4. Alkanesulfonate degradation by novel strains of Achromobacter xylosoxidans, Tsukamurella wratislaviensis and Rhodococcus sp., and evidence for an ethanesulfonate monooxygenase in A. xylosoxidans strain AE4.

    PubMed

    Erdlenbruch, B N; Kelly, D P; Murrell, J C

    2001-12-01

    Novel isolates of Achromobacter xylosoxidans, Tsukamurella wratislaviensis and a Rhodococcus sp. are described. These grew with short-chain alkanesulfonates as their sole source of carbon and energy. T. wratislaviensis strain SB2 grew well with C(3)-C(6) linear alkanesulfonates, isethionate and taurine, Rhodococcus sp. strain CB1 used C(3)-C(10) linear alkanesulfonates, taurine and cysteate, but neither strain grew with ethanesulfonate. In contrast, A. xylosoxidans strain AE4 grew well with ethanesulfonate, making it the first bacterium to be described which can grow with this compound. It also grew with unsubstituted C(3)-C(5) alkanesulfonates and isethionate. Hydrolysis was excluded as a mechanism for alkanesulfonate metabolism in these strains; and evidence is given for a diversity of uptake and desulfonatase systems. We provide evidence for an initial monooxygenase-dependent desulfonation in the metabolism of ethanesulfonate and propanesulfonate by A. xylosoxidans strain AE4.

  5. Isolation of insertion elements from gram-positive Brevibacterium, Corynebacterium and Rhodococcus strains using the Bacillus subtilis sacB gene as a positive selection marker.

    PubMed

    Jäger, W; Schäfer, A; Kalinowski, J; Pühler, A

    1995-02-01

    The sacB gene of Bacillus subtilis was successfully applied in various Arthrobacter, Brevibacterium, Corynebacterium and Rhodococcus strains for the isolation of transposable elements. Three different insertion sequence (IS) elements entrapped in sacB were isolated. The IS elements IS-Bl and IS-Cg isolated from Brevibacterium lactofermentum and Corynebacterium glutamicum, respectively, were found to be similar in size (1.45 kb) and generated target duplications of 8 bp. Their inverted repeats showed homology. In contrast, the IS element IS-Rf isolated from Rhodococcus fascians was only 1.3 kb long and generated a 3-bp target duplication. IS-Cg and IS-Rf were not restricted to their original host strains, and we also found strains harbouring more than one element.

  6. Sequence analysis of the oxidase/reductase genes upstream of the Rhodococcus erythropolis aldehyde dehydrogenase gene thcA reveals a gene organisation different from Mycobacterium tuberculosis.

    PubMed

    Nagy, I; De Mot, R

    1999-01-01

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

  7. Draft Genome Sequence of Rhodococcus erythropolis VSD3, a Diesel Fuel-Degrading and Plant Growth-Promoting Bacterium Isolated from Hedera helix Leaves

    PubMed Central

    Stevens, Vincent; Thijs, Sofie; McAmmond, Breanne; Langill, Tori; Van Hamme, Jonathan; Weyens, Nele

    2017-01-01

    ABSTRACT We report here the 6.55-Mb draft genome sequence of Rhodococcus erythropolis VSD3, a Gram-positive bacterium of the Nocardiaceae family, isolated from leaves of Hedera helix growing at a high-traffic city center in Belgium. The exploration of its genome will contribute to the assessment of its application as an inoculant in phylloremediation approaches. PMID:28232452

  8. Biodegradation of variable-chain-length n-alkanes in Rhodococcus opacus R7 and the involvement of an alkane hydroxylase system in the metabolism.

    PubMed

    Zampolli, Jessica; Collina, Elena; Lasagni, Marina; Di Gennaro, Patrizia

    2014-01-01

    Rhodococcus opacus R7 is a Gram-positive bacterium isolated from a polycyclic aromatic hydrocarbon contaminated soil for its versatile metabolism; indeed the strain is able to grow on naphthalene, o-xylene, and several long- and medium-chain n-alkanes. In this work we determined the degradation of n-alkanes in Rhodococcus opacus R7 in presence of n-dodecane (C12), n-hexadecane (C16), n-eicosane (C20), n-tetracosane (C24) and the metabolic pathway in presence of C12. The consumption rate of C12 was 88%, of C16 was 69%, of C20 was 51% and of C24 it was 78%. The decrement of the degradation rate seems to be correlated to the length of the aliphatic chain of these hydrocarbons. On the basis of the metabolic intermediates determined by the R7 growth on C12, our data indicated that R. opacus R7 metabolizes medium-chain n-alkanes by the primary alcohol formation. This represents a difference in comparison with other Rhodococcus strains, in which a mixture of the two alcohols was observed. By GC-MSD analysis we also identified the monocarboxylic acid, confirming the terminal oxidation. Moreover, the alkB gene cluster from R. opacus R7 was isolated and its involvement in the n-alkane degradation system was investigated by the cloning of this genomic region into a shuttle-vector E. coli-Rhodococcus to evaluate the alkane hydroxylase activity. Our results showed an increased biodegradation of C12 in the recombinant strain R. erythropolis AP (pTipQT1-alkR7) in comparison with the wild type strain R. erythropolis AP. These data supported the involvement of the alkB gene cluster in the n-alkane degradation in the R7 strain.

  9. Draft Genome Sequence of the Endophytic Strain Rhodococcus kyotonensis KB10, a Potential Biodegrading and Antibacterial Bacterium Isolated from Arabidopsis thaliana

    PubMed Central

    Hong, Chi Eun; Jo, Sung Hee

    2016-01-01

    Rhodococcus kyotonensis KB10 is an endophytic bacterium isolated from Arabidopsis thaliana. The organism showed mild antibacterial activity against the phytopathogen Pseudomonas syringae pv. tomato DC3000. This study reports the genome sequence of R. kyotonensis KB10. This bacterium contains an ectoine biosynthesis gene cluster and has the potential to degrade nitroaromatic compounds. The identified bacterium may be a suitable biocontrol agent and degrader of environmental pollutants. PMID:27389269

  10. Members of the genera Paenibacillus and Rhodococcus harbor genes homologous to enterococcal glycopeptide resistance genes vanA and vanB.

    PubMed

    Guardabassi, L; Christensen, H; Hasman, H; Dalsgaard, A

    2004-12-01

    Genes homologous to enterococcal glycopeptide resistance genes vanA and vanB were found in glycopeptide-resistant Paenibacillus and Rhodococcus strains from soil. The putative D-Ala:D-Lac ligase genes in Paenibacillus thiaminolyticus PT-2B1 and Paenibacillus apiarius PA-B2B were closely related to vanA (92 and 87%) and flanked by genes homologous to vanH and vanX in vanA operons.

  11. Members of the Genera Paenibacillus and Rhodococcus Harbor Genes Homologous to Enterococcal Glycopeptide Resistance Genes vanA and vanB

    PubMed Central

    Guardabassi, L.; Christensen, H.; Hasman, H.; Dalsgaard, A.

    2004-01-01

    Genes homologous to enterococcal glycopeptide resistance genes vanA and vanB were found in glycopeptide-resistant Paenibacillus and Rhodococcus strains from soil. The putative d-Ala:d-Lac ligase genes in Paenibacillus thiaminolyticus PT-2B1 and Paenibacillus apiarius PA-B2B were closely related to vanA (92 and 87%) and flanked by genes homologous to vanH and vanX in vanA operons. PMID:15561881

  12. Biodegradation of variable-chain-length n-alkanes in Rhodococcus opacus R7 and the involvement of an alkane hydroxylase system in the metabolism

    PubMed Central

    2014-01-01

    Rhodococcus opacus R7 is a Gram-positive bacterium isolated from a polycyclic aromatic hydrocarbon contaminated soil for its versatile metabolism; indeed the strain is able to grow on naphthalene, o-xylene, and several long- and medium-chain n-alkanes. In this work we determined the degradation of n-alkanes in Rhodococcus opacus R7 in presence of n-dodecane (C12), n-hexadecane (C16), n-eicosane (C20), n-tetracosane (C24) and the metabolic pathway in presence of C12. The consumption rate of C12 was 88%, of C16 was 69%, of C20 was 51% and of C24 it was 78%. The decrement of the degradation rate seems to be correlated to the length of the aliphatic chain of these hydrocarbons. On the basis of the metabolic intermediates determined by the R7 growth on C12, our data indicated that R. opacus R7 metabolizes medium-chain n-alkanes by the primary alcohol formation. This represents a difference in comparison with other Rhodococcus strains, in which a mixture of the two alcohols was observed. By GC-MSD analysis we also identified the monocarboxylic acid, confirming the terminal oxidation. Moreover, the alkB gene cluster from R. opacus R7 was isolated and its involvement in the n-alkane degradation system was investigated by the cloning of this genomic region into a shuttle-vector E. coli-Rhodococcus to evaluate the alkane hydroxylase activity. Our results showed an increased biodegradation of C12 in the recombinant strain R. erythropolis AP (pTipQT1-alkR7) in comparison with the wild type strain R. erythropolis AP. These data supported the involvement of the alkB gene cluster in the n-alkane degradation in the R7 strain. PMID:25401074

  13. Isolation, identification and characterization of a novel Rhodococcus sp. strain in biodegradation of tetrahydrofuran and its medium optimization using sequential statistics-based experimental designs.

    PubMed

    Yao, Yanlai; Lv, Zhenmei; Min, Hang; Lv, Zhenhua; Jiao, Huipeng

    2009-06-01

    Statistics-based experimental designs were applied to optimize the culture conditions for tetrahydrofuran (THF) degradation by a newly isolated Rhodococcus sp. YYL that tolerates high THF concentrations. Single factor experiments were undertaken for determining the optimum range of each of four factors (initial pH and concentrations of K(2)HPO(4).3H(2)O, NH(4)Cl and yeast extract) and these factors were subsequently optimized using the response surface methodology. The Plackett-Burman design was used to identify three trace elements (Mg(2+), Zn(2+)and Fe(2+)) that significantly increased the THF degradation rate. The optimum conditions were found to be: 1.80 g/L NH(4)Cl, 0.81 g/L K(2)HPO(4).3H(2)O, 0.06 g/L yeast extract, 0.40 g/L MgSO(4).7H(2)O, 0.006 g/L ZnSO(4).7H(2)O, 0.024 g/L FeSO(4).7H(2)O, and an initial pH of 8.26. Under these optimized conditions, the maximum THF degradation rate increased to 137.60 mg THF h(-1) g dry weight in Rhodococcus sp. YYL, which was nearly five times of that by the previously described THF degrading Rhodococcus strain.

  14. Purification, cloning, and primary structure of a new enantiomer-selective amidase from a Rhodococcus strain: structural evidence for a conserved genetic coupling with nitrile hydratase.

    PubMed Central

    Mayaux, J F; Cerbelaud, E; Soubrier, F; Yeh, P; Blanche, F; Pétré, D

    1991-01-01

    A new enantiomer-selective amidase active on several 2-aryl propionamides was identified and purified from a newly isolated Rhodococcus strain. The characterized amidase is an apparent homodimer, each molecule of which has an Mr of 48,554; it has a specific activity of 16.5 mumol of S(+)-2-phenylpropionic acid formed per min per mg of enzyme from the racemic amide under our conditions. An oligonucleotide probe was deduced from limited peptide information and was used to clone the corresponding gene, named amdA. As expected, significant homologies were found between the amino acid sequences of the enantiomer-selective amidase of Rhodococcus sp., the corresponding enzyme from Brevibacterium sp. strain R312, and several known amidases, thus confirming the existence of a structural class of amidase enzymes. Genes probably coding for the two subunits of a nitrile hydratase, albeit in an inverse order, were found 39 bp downstream of amdA, suggesting that such a genetic organization might be conserved in different microorganisms. Although we failed to express an active Rhodococcus amidase in Escherichia coli, even in conditions allowing the expression of an active R312 enzyme, the high-level expression of the active recombinant enzyme could be demonstrated in Brevibacterium lactofermentum by using a pSR1-derived shuttle vector. Images FIG. 5 PMID:1938876

  15. Direct and Rapid Analysis of the Adhesion of Bacteria to Solid Surfaces: Interaction of Fluorescently Labeled Rhodococcus Strain GIN-1 (NCIMB 40340) Cells with Titanium-Rich Particles

    PubMed Central

    Fleminger, G.; Shabtai, Y.

    1995-01-01

    A fluorimetric assay which enables direct and accurate analysis of the adhesion of bacteria to solid particles was developed. The assay is based on labeling of the bacteria with fluorescamine, which reacts with primary amino groups on the cell surface to yield a yellow fluorescence that is easily detectable by both fluorescence microscopy and spectrofluorimetry. As an example, fluorescent labeling of Rhodococcus strain GIN-1 (NCIMB 40340) cells enabled the detection and quantitative determination of their adsorption to TiO(inf2) and coal fly ash particles. Exposure of the cells to 10% acetone during the labeling reaction affected neither their viability nor their ability to adhere to these particles. Only a small fraction (^sim2%) of the total cell protein was labeled by fluorescamine upon staining of intact bacterial cells, which may indicate preferential labeling of certain proteins. Specificity studies carried out with the fluorescence assay confirmed previous findings that Rhodococcus strain GIN-1 cells possess high affinities for TiO(inf2), ZnO, and coal fly ash and low affinities for other metal oxides. In principle, the newly developed fluorimetric assay may be used for determination of cell adhesion to any solid matrix by either microscopic examination or epifluorescence measurements. In the present work, the adhesion of several other microorganisms to TiO(inf2) particles was tested as well, but their ability to adhere to these particles was significantly lower than that of Rhodococcus strain GIN-1 cells. PMID:16535188

  16. Direct and Rapid Analysis of the Adhesion of Bacteria to Solid Surfaces: Interaction of Fluorescently Labeled Rhodococcus Strain GIN-1 (NCIMB 40340) Cells with Titanium-Rich Particles.

    PubMed

    Fleminger, G; Shabtai, Y

    1995-12-01

    A fluorimetric assay which enables direct and accurate analysis of the adhesion of bacteria to solid particles was developed. The assay is based on labeling of the bacteria with fluorescamine, which reacts with primary amino groups on the cell surface to yield a yellow fluorescence that is easily detectable by both fluorescence microscopy and spectrofluorimetry. As an example, fluorescent labeling of Rhodococcus strain GIN-1 (NCIMB 40340) cells enabled the detection and quantitative determination of their adsorption to TiO(inf2) and coal fly ash particles. Exposure of the cells to 10% acetone during the labeling reaction affected neither their viability nor their ability to adhere to these particles. Only a small fraction (;sim2%) of the total cell protein was labeled by fluorescamine upon staining of intact bacterial cells, which may indicate preferential labeling of certain proteins. Specificity studies carried out with the fluorescence assay confirmed previous findings that Rhodococcus strain GIN-1 cells possess high affinities for TiO(inf2), ZnO, and coal fly ash and low affinities for other metal oxides. In principle, the newly developed fluorimetric assay may be used for determination of cell adhesion to any solid matrix by either microscopic examination or epifluorescence measurements. In the present work, the adhesion of several other microorganisms to TiO(inf2) particles was tested as well, but their ability to adhere to these particles was significantly lower than that of Rhodococcus strain GIN-1 cells.

  17. Purification, cloning, and primary structure of a new enantiomer-selective amidase from a Rhodococcus strain: structural evidence for a conserved genetic coupling with nitrile hydratase.

    PubMed

    Mayaux, J F; Cerbelaud, E; Soubrier, F; Yeh, P; Blanche, F; Pétré, D

    1991-11-01

    A new enantiomer-selective amidase active on several 2-aryl propionamides was identified and purified from a newly isolated Rhodococcus strain. The characterized amidase is an apparent homodimer, each molecule of which has an Mr of 48,554; it has a specific activity of 16.5 mumol of S(+)-2-phenylpropionic acid formed per min per mg of enzyme from the racemic amide under our conditions. An oligonucleotide probe was deduced from limited peptide information and was used to clone the corresponding gene, named amdA. As expected, significant homologies were found between the amino acid sequences of the enantiomer-selective amidase of Rhodococcus sp., the corresponding enzyme from Brevibacterium sp. strain R312, and several known amidases, thus confirming the existence of a structural class of amidase enzymes. Genes probably coding for the two subunits of a nitrile hydratase, albeit in an inverse order, were found 39 bp downstream of amdA, suggesting that such a genetic organization might be conserved in different microorganisms. Although we failed to express an active Rhodococcus amidase in Escherichia coli, even in conditions allowing the expression of an active R312 enzyme, the high-level expression of the active recombinant enzyme could be demonstrated in Brevibacterium lactofermentum by using a pSR1-derived shuttle vector.

  18. Effect of growth media on cell envelope composition and nitrile hydratase stability in Rhodococcus rhodochrous strain DAP 96253.

    PubMed

    Tucker, Trudy-Ann; Crow, Sidney A; Pierce, George E

    2012-11-01

    Rhodococcus is an important industrial microorganism that possesses diverse metabolic capabilities; it also has a cell envelope, composed of an outer layer of mycolic acids and glycolipids. Selected Rhodococcus species when induced are capable of transforming nitriles to the corresponding amide by the enzyme nitrile hydratase (NHase), and subsequently to the corresponding acid via an amidase. This nitrile biochemistry has generated interest in using the rhodococci as biocatalysts. It was hypothesized that altering sugars in the growth medium might impact cell envelope components and have effects on NHase. When the primary carbon source in growth media was changed from glucose to fructose, maltose, or maltodextrin, the NHase activity increased. Cells grown in the presence of maltose and maltodextrin showed the highest activities against propionitrile, 197 and 202 units/mg cdw, respectively. Stability of NHase was also affected as cells grown in the presence of maltose and maltodextrin retained more NHase activity at 55 °C (45 and 23 %, respectively) than cells grown in the presence of glucose or fructose (19 and 10 %, respectively). Supplementation of trehalose in the growth media resulted in increased NHase stability at 55 °C, as cells grown in the presence of glucose retained 40 % NHase activity as opposed to 19 % without the presence of trehalose. Changes in cell envelope components, such mycolic acids and glycolipids, were evaluated by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC), respectively. Changing sugars and the addition of inducing components for NHase, such as cobalt and urea in growth media, resulted in changes in mycolic acid profiles. Mycolic acid content increased 5 times when cobalt and urea were added to media with glucose. Glycolipids levels were also affected by the changes in sugars and addition of inducing components. This research demonstrates that carbohydrate selection impacts NHase activity and

  19. Electrical resistivity tomography to monitor enhanced biodegradation of hydrocarbons with Rhodococcus erythropolis T902.1 at a pilot scale.

    PubMed

    Masy, Thibaut; Caterina, David; Tromme, Olivier; Lavigne, Benoît; Thonart, Philippe; Hiligsmann, Serge; Nguyen, Frédéric

    2016-01-01

    Petroleum hydrocarbons (HC) represent the most widespread contaminants and in-situ bioremediation remains a competitive treatment in terms of cost and environmental concerns. However, the efficiency of such a technique (by biostimulation or bioaugmentation) strongly depends on the environment affected and is still difficult to predict a priori. In order to overcome these uncertainties, Electrical Resistivity Tomography (ERT) appears as a valuable non-invasive tool to detect soil heterogeneities and to monitor biodegradation. The main objective of this study was to isolate an electrical signal linked to an enhanced bacterial activity with ERT, in an aged HC-contaminated clay loam soil. To achieve this, a pilot tank was built to mimic field conditions. Compared to a first insufficient biostimulation phase, bioaugmentation with Rhodococcus erythropolis T902.1 led to a HC depletion of almost 80% (6900 to 1600ppm) in 3months in the center of the contaminated zone, where pollutants were less bioavailable. In the meantime, lithological heterogeneities and microbial activities (growth and biosurfactant production) were successively discriminated by ERT images. In the future, this cost-effective technique should be more and more transferred to the field in order to monitor biodegradation processes and assist in selecting the most appropriate remediation technique. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Electrical resistivity tomography to monitor enhanced biodegradation of hydrocarbons with Rhodococcus erythropolis T902.1 at a pilot scale

    NASA Astrophysics Data System (ADS)

    Masy, Thibaut; Caterina, David; Tromme, Olivier; Lavigne, Benoît; Thonart, Philippe; Hiligsmann, Serge; Nguyen, Frédéric

    2016-01-01

    Petroleum hydrocarbons (HC) represent the most widespread contaminants and in-situ bioremediation remains a competitive treatment in terms of cost and environmental concerns. However, the efficiency of such a technique (by biostimulation or bioaugmentation) strongly depends on the environment affected and is still difficult to predict a priori. In order to overcome these uncertainties, Electrical Resistivity Tomography (ERT) appears as a valuable non-invasive tool to detect soil heterogeneities and to monitor biodegradation. The main objective of this study was to isolate an electrical signal linked to an enhanced bacterial activity with ERT, in an aged HC-contaminated clay loam soil. To achieve this, a pilot tank was built to mimic field conditions. Compared to a first insufficient biostimulation phase, bioaugmentation with Rhodococcus erythropolis T902.1 led to a HC depletion of almost 80% (6900 to 1600 ppm) in 3 months in the center of the contaminated zone, where pollutants were less bioavailable. In the meantime, lithological heterogeneities and microbial activities (growth and biosurfactant production) were successively discriminated by ERT images. In the future, this cost-effective technique should be more and more transferred to the field in order to monitor biodegradation processes and assist in selecting the most appropriate remediation technique.

  1. Functional characterization and stability improvement of a ‘thermophilic-like’ ene-reductase from Rhodococcus opacus 1CP

    PubMed Central

    Riedel, Anika; Mehnert, Marika; Paul, Caroline E.; Westphal, Adrie H.; van Berkel, Willem J. H.; Tischler, Dirk

    2015-01-01

    Ene-reductases (ERs) are widely applied for the asymmetric synthesis of relevant industrial chemicals. A novel ER OYERo2 was found within a set of 14 putative old yellow enzymes (OYEs) obtained by genome mining of the actinobacterium Rhodococcus opacus 1CP. Multiple sequence alignment suggested that the enzyme belongs to the group of ‘thermophilic-like’ OYEs. OYERo2 was produced in Escherichia coli and biochemically characterized. The enzyme is strongly NADPH dependent and uses non-covalently bound FMNH2 for the reduction of activated α,β-unsaturated alkenes. In the active form OYERo2 is a dimer. Optimal catalysis occurs at pH 7.3 and 37°C. OYERo2 showed highest specific activities (45-50 U mg-1) on maleimides, which are efficiently converted to the corresponding succinimides. The OYERo2-mediated reduction of prochiral alkenes afforded the (R)-products with excellent optical purity (ee > 99%). OYERo2 is not as thermo-resistant as related OYEs. Introduction of a characteristic intermolecular salt bridge by site-specific mutagenesis raised the half-life of enzyme inactivation at 32°C from 28 to 87 min and improved the tolerance toward organic co-solvents. The suitability of OYERo2 for application in industrial biocatalysis is discussed. PMID:26483784

  2. Transfer of the virulence-associated protein a-bearing plasmid between field strains of virulent and avirulent Rhodococcus equi.

    PubMed

    Stoughton, W; Poole, T; Kuskie, K; Liu, M; Bishop, K; Morrissey, A; Takai, S; Cohen, N

    2013-01-01

    Virulent and avirulent isolates of Rhodococcus equi coexist in equine feces and the environment and are a source of infection for foals. The extent to which plasmid transfer occurs among field strains is ill-defined and this information is important for understanding the epidemiology of R. equi infections of foals. To estimate the frequency of transfer of the virulence plasmid between virulent and avirulent strains of R. equi derived from foals and their environment. None. In vitro study; 5 rifampin-susceptible, virulent R. equi isolates obtained from clinically affected foals or air samples from a farm with a history of recurrent R. equi foal pneumonia were each mixed with 5 rifampin-resistant, avirulent isolates derived from soil samples, using solid medium, at a ratio of 10 donor cells (virulent) per recipient cell. Presumed transconjugates were detected by plating on media with rifampin and colony immunoblotting to detect the presence of the virulence-associated protein A. Three presumed transconjugates were detected among 2,037 recipient colonies, indicating an overall estimated transfer frequency of 0.15% (95% CI, 0.03–0.43%). All 3 transconjugates were associated with a single donor and 2 recipient strains. Genotyping and multiplex PCR of presumed transconjugates demonstrated transfer of the virulence-associated protein A-bearing plasmid between virulent and avirulent R. equi. Transfer of the virulence plasmid occurs with relatively high frequency. These findings could impact strategies to control or prevent R. equi through environmental management.

  3. Identification of pathogens and virulence profile of Rhodococcus equi and Escherichia coli strains obtained from sand of parks.

    PubMed

    Fernandes, M C; Takai, S; Leite, D S; Pinto, J P A N; Brandão, P E; Santarém, V A; Listoni, F J P; Da Silva, A V; Ribeiro, M G

    2013-01-01

    The identification of pathogens of viral (Rotavirus, Coronavirus), parasitic (Toxocara spp.) and bacterial (Escherichia coli, Salmonella spp., Rhodococcus equi) origin shed in feces, and the virulence profile of R. equi and E. coli isolates were investigated in 200 samples of sand obtained from 40 parks, located in central region of state of Sao Paulo, Brazil, using different diagnostic methods. From 200 samples analyzed, 23 (11.5%) strains of R. equi were isolated. None of the R. equi isolates showed a virulent (vapA gene) or intermediately virulent (vapB gene) profiles. Sixty-three (31.5%) strains of E. coli were identified. The following genes encoding virulence factors were identified in E. coli: eae, bfp, saa, iucD, papGI, sfa and hly. Phylogenetic classification showed that 63 E. coli isolates belonged to groups B1 (52.4%), A (25.4%) and B2 (22.2%). No E. coli serotype O157:H7 was identified. Eggs of Toxocara sp. were found in three parks and genetic material of bovine Coronavirus was identified in one sample of one park. No Salmonella spp. and Rotavirus isolates were identified in the samples of sand. The presence of R. equi, Toxocara sp, bovine Coronavirus and virulent E. coli isolates in the environment of parks indicates that the sanitary conditions of the sand should be improved in order to reduce the risks of fecal transmission of pathogens of zoonotic potential to humans in these places.

  4. Association of perinatal exposure to airborne Rhodococcus equi with risk of pneumonia caused by R equi in foals.

    PubMed

    Cohen, Noah D; Chaffin, M Keith; Kuskie, Kyle R; Syndergaard, Melissa K; Blodgett, Glenn P; Takai, Shinji

    2013-01-01

    To determine whether the concentrations of airborne virulent Rhodococcus equi in stalls housing foals during the first 2 weeks after birth are associated with subsequent development of R equi pneumonia in those foals. Air samples collected from foaling stalls and holding pens in which foals were housed during the first 2 weeks after birth. At a breeding farm in Texas, air samples (500 L each) were collected (January through May 2011) from stalls and pens in which 121 foals were housed on day 1 and on days 4, 7, and 14 after birth. For each sample, the concentration of airborne virulent R equi was determined with an immunoblot technique. The association between development of pneumonia and airborne R equi concentration was evaluated via random-effects Poisson regression analysis. Some air samples were not available for analysis. Of the 471 air samples collected from stalls that housed 121 foals, 90 (19%) contained virulent R equi. Twenty-four of 121 (20%) foals developed R equi pneumonia. Concentrations of virulent R equi in air samples from stalls housing foals that developed R equi pneumonia were significantly higher than those in samples from stalls housing foals that did not develop pneumonia. Accounting for disease effects, air sample concentrations of virulent R equi did not differ significantly by day after birth or by month of birth. Exposure of foals to airborne virulent R equi during the first 2 weeks after birth was significantly (and likely causally) associated with development of R equi pneumonia.

  5. Bronchopneumonia in wild boar (Sus scrofa) caused by Rhodococcus equi carrying the VapB type 8 plasmid.

    PubMed

    de Vargas, Agueda Castagna; Monego, Fernanda; Gressler, Letícia Trevisan; de Avila Botton, Sônia; Lazzari, Andrea Maria; da Costa, Mateus Matiuzzi; Ecco, Roselene; Ribeiro, Márcio Garcia; Lara, Gustavo Henrique Batista; Takai, Shinji

    2013-03-25

    Rhodococcus equi is associated with pyogranulomatous infections, especially in foals, and this bacterium has also emerged as a pathogen for humans, particularly immunocompromised patients. R. equi infections in pigs, wild boar (Sus scrofa) and humans are mainly due to strains carrying the intermediate virulence (VapB) plasmid. In Brazil, R. equi carrying the VapB type 8 plasmid is the most common type recovered from humans co-infected with the human immunodeficiency virus (HIV). R. equi infection in pigs and wild boar is restricted predominantly to the lymphatic system, without any reports of pulmonary manifestations. This report describes the microbiological and histopathological findings, and molecular characterization of R. equi in two bronchopneumonia cases in wild boar using PCR and plasmid profile analysis by digestion with restriction endonucleases. The histological findings were suggestive of pyogranulomatous infection, and the plasmid profile of both R. equi isolates enabled the characterization of the strains as VapB type 8. This is the first report of bronchopneumonia in wild boar due to R. equi. The detection of the VapB type 8 plasmid in R. equi isolates emphasize that wild boar may be a potential source of pathogenic R. equi strains for humans.

  6. Unsaturated fatty acids as modulators of macrophage respiratory burst in the immune response against Rhodococcus equi and Pseudomonas aeruginosa.

    PubMed

    Adolph, Stephanie; Schoeniger, Axel; Fuhrmann, Herbert; Schumann, Julia

    In this paper, using the monocyte/macrophage cell line RAW264.7, we systematically investigate the impact of macrophage enrichment with unsaturated fatty acids on cellular radical synthesis. We found that the intracellular production of reactive nitrogen and oxygen intermediates depends on the activation status of the macrophages. For unstimulated macrophages PUFA enrichment resulted in an increase in cellular radical synthesis. For stimulated macrophages, instead, an impeding action of unsaturated fatty acids on the respiratory burst could be seen. Of particular importance, the impact of unsaturated fatty acids on the macrophage respiratory burst was also observed in RAW264.7 cells cocultivated with viable bacteria of the species Rhodococcus equi or Pseudomonas aeruginosa. PUFA supplementation of macrophages in the presence of R. equi or P. aeruginosa reduced the pathogen-stimulated synthesis of reactive nitrogen and oxygen intermediates. Furthermore, the unsaturated fatty acids were found to impede the expression of the myeloperoxidase gene and to reduce the activity of the enzyme. Hence, our data provide indications of a possible value of PUFA application to people suffering from chronic infections with R. equi and P. aeruginosa as a concomitant treatment to attenuate an excessive respiratory burst. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Comparative transcriptomics elucidates adaptive phenol tolerance and utilization in lipid-accumulating Rhodococcus opacus PD630

    SciTech Connect

    Yoneda, Aki; Henson, William R.; Goldner, Nicholas K.; Park, Kun Joo; Forsberg, Kevin J.; Kim, Soo Ji; Pesesky, Mitchell W.; Foston, Marcus; Dantas, Gautam; Moon, Tae Seok

    2016-02-02

    Lignin-derived (e.g. phenolic) compounds can compromise the bioconversion of lignocellulosic biomass to fuels and chemicals due to their toxicity and recalcitrance. The lipid-accumulating bacterium Rhodococcus opacus PD630 has recently emerged as a promising microbial host for lignocellulose conversion to value-added products due to its natural ability to tolerate and utilize phenolics. To gain a better understanding of its phenolic tolerance and utilization mechanisms, we adaptively evolved R. opacus over 40 passages using phenol as its sole carbon source (up to 373% growth improvement over wild-type), and extensively characterized two strains from passages 33 and 40. The two adapted strains showed higher phenol consumption rates (~20 mg/l/h) and ~2-fold higher lipid production from phenol than the wild-type strain.Whole-genome sequencing and comparative transcriptomics identified highly-upregulated degradation pathways and putative transporters for phenol in both adapted strains, highlighting the important linkage between mechanisms of regulated phenol uptake, utilization, and evolved tolerance. Our study shows that the R. opacus mutants are likely to use their transporters to import phenol rather than export them, suggesting a new aromatic tolerance mechanism. The identified tolerance genes and pathways are promising candidates for future metabolic engineering in R. opacus for improved lignin conversion to lipid-based products.

  8. Limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14 belongs to a novel class of epoxide hydrolases.

    PubMed

    van der Werf, M J; Overkamp, K M; de Bont, J A

    1998-10-01

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

  9. Regulation of plasmid-encoded isoprene metabolism in Rhodococcus, a representative of an important link in the global isoprene cycle

    PubMed Central

    Crombie, Andrew T; Khawand, Myriam El; Rhodius, Virgil A; Fengler, Kevin A; Miller, Michael C; Whited, Gregg M; McGenity, Terry J; Murrell, J Colin

    2015-01-01

    Emissions of biogenic volatile organic compounds (VOCs) form an important part of the global carbon cycle, comprising a significant proportion of net ecosystem productivity. They impact atmospheric chemistry and contribute directly and indirectly to greenhouse gases. Isoprene, emitted largely from plants, comprises one third of total VOCs, yet in contrast to methane, which is released in similar quantities, we know little of its biodegradation. Here, we report the genome of an isoprene degrading isolate, Rhodococcus sp. AD45, and, using mutagenesis shows that a plasmid-encoded soluble di-iron centre isoprene monooxygenase (IsoMO) is essential for isoprene metabolism. Using RNA sequencing (RNAseq) to analyse cells exposed to isoprene or epoxyisoprene in a substrate-switch time-course experiment, we show that transcripts from 22 contiguous genes, including those encoding IsoMO, were highly upregulated, becoming among the most abundant in the cell and comprising over 25% of the entire transcriptome. Analysis of gene transcription in the wild type and an IsoMO-disrupted mutant strain showed that epoxyisoprene, or a subsequent product of isoprene metabolism, rather than isoprene itself, was the inducing molecule. We provide a foundation of molecular data for future research on the environmental biological consumption of this important, climate-active compound. PMID:25727256

  10. Enantioselective Metabolism of Chiral 3-Phenylbutyric Acid, an Intermediate of Linear Alkylbenzene Degradation, by Rhodococcus rhodochrous PB1

    PubMed Central

    Simoni, S.; Klinke, S.; Zipper, C.; Angst, W.; Kohler, H. E.

    1996-01-01

    Rhodococcus rhodochrous PB1 was isolated from compost soil by selective culture with racemic 3-phenylbutyric acid as the sole carbon and energy source. Growth experiments with the single pure enantiomers as well as with the racemate showed that only one of the two enantiomers, (R)-3-phenylbutyric acid, supported growth of strain PB1. Nevertheless, (S)-3-phenylbutyric acid was cometabolically transformed to, presumably, (S)-3-(2,3-dihydroxyphenyl)butyric acid (the absolute configuration at the C-3 atom is not known yet) by (R)-3-phenylbutyric acid-grown cells of strain PB1, as shown by (sup1)H nuclear magnetic resonance spectroscopy of the partially purified compound and gas chromatography-mass spectrometry analysis of the trimethylsilyl derivative. Oxygen uptake rates suggest that either 3-phenylpropionic acid or cinnamic acid (trans-3-phenyl-2-propenoic acid) is the substrate for aromatic ring hydroxylation. This view is substantiated by the fact that 3-(2,3-dihydroxyphenyl)propionic acid was a substrate for meta cleavage in cell extracts of (R)-3-phenylbutyric acid-grown cells of strain PB1. Gas chromatography-mass spectrometry analysis of trimethylsilane-treated ethyl acetate extracts of incubation mixtures showed that both the meta-cleavage product, 2-hydroxy-6-oxo-2,4-nonadiene-1,9-dicarboxylic acid, and succinate, a hydrolysis product thereof, were formed during such incubations. PMID:16535265

  11. Production of carotenoids and lipids by Rhodococcus opacus PD630 in batch and fed-batch culture.

    PubMed

    Thanapimmetha, Anusith; Suwaleerat, Tharatron; Saisriyoot, Maythee; Chisti, Yusuf; Srinophakun, Penjit

    2017-01-01

    Production of carotenoids by Rhodococcus opacus PD630 is reported. A modified mineral salt medium formulated with glycerol as an inexpensive carbon source was used for the fermentation. Ammonium acetate was the nitrogen source. A dry cell mass concentration of nearly 5.4 g/L could be produced in shake flasks with a carotenoid concentration of 0.54 mg/L. In batch culture in a 5 L bioreactor, without pH control, the maximum dry biomass concentration was ~30 % lower than in shake flasks and the carotenoids concentration was 0.09 mg/L. Both the biomass concentration and the carotenoids concentration could be raised using a fed-batch operation with a feed mixture of ammonium acetate and acetic acid. With this strategy, the final biomass concentration was 8.2 g/L and the carotenoids concentration was 0.20 mg/L in a 10-day fermentation. A control of pH proved to be unnecessary for maximizing the production of carotenoids in this fermentation.

  12. [Function analysis of the effective strain Rhodococcus ruber Em1 in wastewater treatment system by quantitative competitive PCR].

    PubMed

    Huang, Ling; Li, Xi-wu; Li, Xu-dong; Liu, Shuang-jiang; Liu, Zhi-pei; Tan, Zhou-liang

    2007-04-01

    A quantitative competitive PCR (QC-PCR) system was developed to quantify the number and analyze the function of the Rhodococcus ruber Em1 strain in a wastewater treatment system in Nanchong oil refinery plant. Strain Em1 was able to degrade various kinds of hydrocarbons and aromatic compounds with high efficiency and produce bioemulsifier, so it was introduced into the waste liquid petroleum-disposing system. The sediment samples were collected from the disposing system in the range of 5 months, and then the numbers of strain Eml and degrading efficiencies were studied. The results showed that the primers based on 16S rRNA gene sequence of strain Em1 were specific at species level. The PCR products amplified from sediment total DNA with the specific primers were cloned and sequenced, in which 62.2% were the fragments of 16S rRNA gene of strain Em1. Furthermore, the number of Em1 strain ranging from 3.4 x 10(5) - 4.3 x 10(8) CFU/g in the sediment samples were detected, which indicated that the strain Eml added into purposely did exist stably and reproduced well in the waste-deposing system during a long period. The high relativity, with relative coefficient R2 of 0.89, between Eml cell number and the amount of COD (Chemical Oxygen Demand) removal proved that the strain Em1 played an important role in this bio-augmentation treatment system.

  13. Acrylamide synthesis using agar entrapped cells of Rhodococcus rhodochrous PA-34 in a partitioned fed batch reactor.

    PubMed

    Raj, Jog; Sharma, Nitya Nand; Prasad, Shreenath; Bhalla, Tek Chand

    2008-01-01

    The nitrile hydratase (Nhase) induced cells of Rhodococcus rhodochrous PA-34 catalyzed the conversion of acrylonitrile to acrylamide. The cells of R. rhodochrous PA-34 immobilized in 2% (w/v) agar (1.76 mg dcw/ml agar matrix) exhibited maximum Nhase activity (8.25 U/mg dcw) for conversion of acrylonitrile to acrylamide at 10 degrees C in the reaction mixture containing 0.1 M potassium phosphate buffer (pH 7.5), 8% (w/v) acrylonitrile and immobilized cells equivalent to 1.12 mg dcw (dry cell weight) per ml. In a partitioned fed batch reaction at 10 degrees C, using 1.12 g dcw immobilized cells in a final volume of 1 l, a total of 372 g of acrylonitrile was completely hydrated to acrylamide (498 g) in 24 h. From the above reaction mixture 87% acrylamide (432 g) was recovered through crystallization at 4 degrees C. By recycling the immobilized biocatalyst (six times), a total of 2,115 g acrylamide was produced.

  14. Purification, crystallization and preliminary X-ray crystallographic analysis of 3-ketosteroid Δ1-dehydrogenase from Rhodococcus erythropolis SQ1

    PubMed Central

    Rohman, Ali; van Oosterwijk, Niels; Dijkstra, Bauke W.

    2012-01-01

    3-Ketosteroid Δ1-dehydrogenase plays a crucial role in the early steps of steroid degradation by introducing a double bond between the C1 and C2 atoms of the A-ring of its 3-ketosteroid substrates. The 3-ketosteroid Δ1-dehydrogenase from Rhodococcus erythropolis SQ1, a 56 kDa flavoprotein, was crystallized using the sitting-drop vapour-diffusion method at room temperature. The crystals grew in various buffers over a wide pH range (from pH 5.5 to 10.5), but the best crystallization condition consisted of 2%(v/v) PEG 400, 0.1 M HEPES pH 7.5, 2.0 M ammonium sulfate. A native crystal diffracted X-rays to 2.0 Å resolution. It belonged to the primitive orthorhombic space group P212121, with unit-cell parameters a = 107.4, b = 131.6, c = 363.2 Å, and contained eight molecules in the asymmetric unit. The initial structure of the enzyme was solved using multi-wavelength anomalous dispersion (MAD) data collected from a Pt-derivatized crystal. PMID:22691786

  15. The Equine Antimicrobial Peptide eCATH1 Is Effective against the Facultative Intracellular Pathogen Rhodococcus equi in Mice

    PubMed Central

    Schlusselhuber, Margot; Torelli, Riccardo; Martini, Cecilia; Leippe, Matthias; Cattoir, Vincent; Leclercq, Roland; Laugier, Claire; Grötzinger, Joachim; Sanguinetti, Maurizio

    2013-01-01

    Rhodococcus equi, the causal agent of rhodococcosis, is a major pathogen of foals and is also responsible for severe infections in immunocompromised humans. Of great concern, strains resistant to currently used antibiotics have emerged. As the number of drugs that are efficient in vivo is limited because of the intracellular localization of the bacterium inside macrophages, new active but cell-permeant drugs will be needed in the near future. In the present study, we evaluated, by in vitro and ex vivo experiments, the ability of the alpha-helical equine antimicrobial peptide eCATH1 to kill intracellular bacterial cells. Moreover, the therapeutic potential of the peptide was assessed in experimental rhodococcosis induced in mice, while the in vivo toxicity was evaluated by behavioral and histopathological analysis. The study revealed that eCATH1 significantly reduced the number of bacteria inside macrophages. Furthermore, the bactericidal potential of the peptide was maintained in vivo at doses that appeared to have no visible deleterious effects for the mice even after 7 days of treatment. Indeed, daily subcutaneous injections of 1 mg/kg body weight of eCATH1 led to a significant reduction of the bacterial load in organs comparable to that obtained after treatment with 10 mg/kg body weight of rifampin. Interestingly, the combination of the peptide with rifampin showed a synergistic interaction in both ex vivo and in vivo experiments. These results emphasize the therapeutic potential that eCATH1 represents in the treatment of rhodococcosis. PMID:23817377

  16. Evaluation of biosurfactants grown in corn oil by Rhodococcus rhodochrous on removing of heavy metal ion from aqueous solution

    SciTech Connect

    Suryanti, Venty Hastuti, Sri; Pujiastuti, Dwi

    2016-02-08

    The potential application of biosurfactants to remove heavy metal ion from aqueous solution by batch technique was examined. The glycolipids type biosurfactants were grown in a media containing of 20% v/v corn oil with 7 days of fermentation by Rhodococcus rhodochrous. The biosurfactants reduced the surface tension of water of about 51% from 62 mN/m to 30 mN/m. The biosurfactant increased the E24 of water-palm oil emulsion of about 55% from 43% to 97% and could maintain this E24 value of above 50% for up to 9 days. Heavy metal ion removal, in this case cadmium ion, by crude and patially purified biosurfactants has been investigated from aqueous solution at pH 6. Adsorption capacity of Cd(II) ion by crude biosurfactant with 5 and 10 minutes of contact times were 1.74 and 1.82 mg/g, respectively. Additionally, the adsorption capacity of Cd(II) ion by partially purified biosurfactant with 5 and 10 minutes of contact times were 0.79 and 1.34 mg/g, respectively. The results demonstrated that the adsorption capacity of Cd(II) ion by crude biosurfactant was higher than that of by partially purified biosurfactant. The results suggested that the biosurfactant could be used in the removal of heavy metal ions from aqueous solution.

  17. Expression control of nitrile hydratase and amidase genes in Rhodococcus erythropolis and substrate specificities of the enzymes.

    PubMed

    Rucká, Lenka; Volkova, Olga; Pavlík, Adam; Kaplan, Ondřej; Kracík, Martin; Nešvera, Jan; Martínková, Ludmila; Pátek, Miroslav

    2014-06-01

    Bacterial amidases and nitrile hydratases can be used for the synthesis of various intermediates and products in the chemical and pharmaceutical industries and for the bioremediation of toxic pollutants. The aim of this study was to analyze the expression of the amidase and nitrile hydratase genes of Rhodococcus erythropolis and test the stereospecific nitrile hydratase and amidase activities on chiral cyanohydrins. The nucleotide sequences of the gene clusters containing the oxd (aldoxime dehydratase), ami (amidase), nha1, nha2 (subunits of the nitrile hydratase), nhr1, nhr2, nhr3 and nhr4 (putative regulatory proteins) genes of two R. erythropolis strains, A4 and CCM2595, were determined. All genes of both of the clusters are transcribed in the same direction. RT-PCR analysis, primer extension and promoter fusions with the gfp reporter gene showed that the ami, nha1 and nha2 genes of R. erythropolis A4 form an operon transcribed from the Pami promoter and an internal Pnha promoter. The activity of Pami was found to be weakly induced when the cells grew in the presence of acetonitrile, whereas the Pnha promoter was moderately induced by both the acetonitrile or acetamide used instead of the inorganic nitrogen source. However, R. erythropolis A4 cells showed no increase in amidase and nitrile hydratase activities in the presence of acetamide or acetonitrile in the medium. R. erythropolis A4 nitrile hydratase and amidase were found to be effective at hydrolysing cyanohydrins and 2-hydroxyamides, respectively.

  18. Purification and characterization of a novel nitrilase of Rhodococcus rhodochrous K22 that acts on aliphatic nitriles.

    PubMed Central

    Kobayashi, M; Yanaka, N; Nagasawa, T; Yamada, H

    1990-01-01

    A novel nitrilase that preferentially catalyzes the hydrolysis of aliphatic nitriles to the corresponding carboxylic acids and ammonia was found in the cells of a facultative crotononitrile-utilizing actinomycete isolated from soil. The strain was taxonomically studied and identified as Rhodococcus rhodochrous. The nitrilase was purified, with 9.08% overall recovery, through five steps from a cell extract of the stain. After the last step, the purified enzyme appeared to be homogeneous, as judged by polyacrylamide gel electrophoresis, analytical centrifugation, and double immunodiffusion in agarose. The relative molecular weight values for the native enzyme, estimated from the ultracentrifugal equilibrium and by high-performance liquid chromatography, were approximately 604,000 +/- 30,000 and 650,000, respectively, and the enzyme consisted of 15 to 16 subunits identical in molecular weight (41,000). The enzyme acted on aliphatic olefinic nitriles such as crotononitrile and acrylonitrile as the most suitable substrates. The apparent Km values for crotononitrile and acrylonitrile were 18.9 and 1.14 mM, respectively. The nitrilase also catalyzed the direct hydrolysis of saturated aliphatic nitriles, such as valeronitrile, 4-chlorobutyronitrile, and glutaronitrile, to the corresponding acids without the formation of amide intermediates. Hence, the R. rhodochrous K22 nitrilase is a new type distinct from all other nitrilases that act on aromatic and related nitriles. Images PMID:2394676

  19. Inhibition of diethyl ether degradation in Rhodococcus sp. strain DEE5151 by glutaraldehyde and ethyl vinyl ether.

    PubMed

    Kim, Yong-Hak; Engesser, Karl-Heinrich

    2005-02-15

    Alkyl ether-degrading Rhodococcus sp. strain DEE5151, isolated from activated sewage sludge, has an activity for the oxidation of a variety of alkyl ethers, aralkyl ethers and dibenzyl ether. The whole cell activity for diethyl ether oxidation was effectively inhibited by 2,3-dihydrofurane, ethyl vinyl ether and glutaraldehyde. Glutaraldehyde of less than 30 microM inhibited the activity by a competitive manner with the inhibition constant, K(I) of 7.07+/-1.36 microM. The inhibition type became mixed at higher glutaraldehyde concentrations >30 microM, probably due to the inactivation of the cell activity by the Schiff-base formation. Structurally analogous ethyl vinyl ether inhibited the diethyl ether oxidation activity in a mixed manner with decreasing the apparent maximum oxidation rate, v(max)(app), and increasing the apparent Michaelis-Menten constant, K(M)(app). The mixed type inhibition by ethyl vinyl ether seemed to be introduced not only by the structure similarity with diethyl ether, but also by the reactivity of the vinyl ether with cellular components in the whole cell system.

  20. Fasciation induction by the phytopathogen Rhodococcus fascians depends upon a linear plasmid encoding a cytokinin synthase gene.

    PubMed

    Crespi, M; Messens, E; Caplan, A B; van Montagu, M; Desomer, J

    1992-03-01

    Rhodococcus fascians is a nocardiform bacteria that induces leafy galls (fasciation) on dicotyledonous and several monocotyledonous plants. The wild-type strain D188 contained a conjugative, 200 kb linear extrachromosomal element, pFiD188. Linear plasmid-cured strains were avirulent and reintroduction of this linear element restored virulence. Pulsed field electrophoresis indicated that the chromosome might also be a linear molecule of 4 megabases. Three loci involved in phytopathogenicity have been identified by insertion mutagenesis of this Fi plasmid. Inactivation of the fas locus resulted in avirulent strains, whereas insertions in the two other loci affected the degree of virulence, yielding attenuated (att) and hypervirulent (hyp) bacteria. One of the genes within the fas locus encoded an isopentenyltranferase (IPT) with low homology to analogous proteins from Gram-negative phytopathogenic bacteria. IPT activity was detected after expression of this protein in Escherichia coli cells. In R.fascians, ipt expression could only be detected in bacteria induced with extracts from fasciated tissue. R.fascians strains without the linear plasmid but containing this fas locus alone could not provoke any phenotype on plants, indicating additional genes from the linear plasmid were also essential for virulence. These studies, the first genetic analysis of the interaction of a Gram-positive bacterium with plants, suggest that a novel mechanism for plant tumour induction has evolved in R.fascians independently from the other branches of the eubacteria.

  1. The phytopathogen Rhodococcus fascians breaks apical dominance and activates axillary meristems by inducing plant genes involved in hormone metabolism.

    PubMed

    Simón-Mateo, Carmen; Depuydt, Stephen; DE Oliveira Manes, Carmem Lara; Cnudde, Filip; Holsters, Marcelle; Goethals, Koen; Vereecke, Danny

    2006-03-01

    SUMMARY Rhodococcus fascians is a Gram-positive bacterium that interacts with many plant species and induces multiple shoots through a combination of activation of dormant axillary meristems and de novo meristem formation. Although phenotypic analysis of the symptoms of infected plants clearly demonstrates a disturbance of the phytohormonal balance and an activation of the cell cycle, the actual mechanism of symptom development and the targets of the bacterial signals are unknown. To elucidate the molecular pathways that are responsive to R. fascians infection, differential display was performed on Nicotiana tabacum as a host. Four differentially expressed genes could be identified that putatively encode a senescence-associated protein, a gibberellin 2-oxidase, a P450 monooxygenase and a proline dehydrogenase. The differential expression of the three latter genes was confirmed on infected Arabidopsis thaliana plants by quantitative reverse transcription polymerase chain reactions, supporting their general function in R. fascians-induced symptom development. The role of these genes in hormone metabolism, especially of gibberellin and abscisic acid, in breaking apical dominance and in activating axillary meristems, which are processes associated with symptom development, is discussed.

  2. Bacterial and plant signal integration via D3-type cyclins enhances symptom development in the Arabidopsis-Rhodococcus fascians interaction.

    PubMed

    Stes, Elisabeth; Biondi, Stefania; Holsters, Marcelle; Vereecke, Danny

    2011-06-01

    The phytopathogenic actinomycete Rhodococcus fascians drives its host to form a nutrient-rich niche by secreting a mixture of cytokinins that triggers plant cell division and shoot formation. The discrepancy between the relatively low amount of secreted cytokinins and the severe impact of R. fascians infection on plant development has puzzled researchers for a long time. Polyamine and transcript profiling of wild-type and cytokinin receptor mutant plants revealed that the bacterial cytokinins directly stimulated the biosynthesis of plant putrescine by activating arginine decarboxylase expression. Pharmacological experiments showed that the increased levels of putrescine contributed to the severity of the symptoms. Thus, putrescine functions as a secondary signal that impinges on the cytokinin-activated pathway, amplifying the hormone-induced changes that lead to the formation of a leafy gall. Exogenous putrescine and treatment with polyamine biosynthesis inhibitors combined with transcript and polyamine analyses of wild-type and mutant plants indicated that the direct target of both the bacterial cytokinins and plant putrescine was the expression of D3-type cyclins. Hence, the activated d-type cyclin/retinoblastoma/E2F transcription factor pathway integrates both external and internal hormonal signals, stimulating mitotic cell divisions and inducing pathological plant organogenesis.

  3. Leafy gall formation is controlled by fasR, an AraC-type regulatory gene in Rhodococcus fascians.

    PubMed

    Temmerman, W; Vereecke, D; Dreesen, R; Van Montagu, M; Holsters, M; Goethals, K

    2000-10-01

    Rhodococcus fascians can interact with many plant species and induce the formation of either leafy galls or fasciations. To provoke symptoms, R. fascians strain D188 requires pathogenicity genes that are located on a linear plasmid, pFiD188. The fas genes are essential for virulence and constitute an operon that encodes, among other functions, a cytokinin synthase gene. Expression of the fas genes is induced by extracts of infected plant tissue only. We have isolated an AraC-type regulatory gene, fasR, located on pFiD188, which is indispensable for pathogenesis and for fas gene expression. The combined results of our experiments show that in vitro expression of the fas genes in a defined medium is strictly regulated and that several environmental factors (pH, carbon and nitrogen sources, phosphate and oxygen content, and cell density) and regulatory proteins are involved. We further show that expression of the fas genes is controlled at both the transcriptional and the translational levels. The complex expression pattern probably reflects the necessity of integrating a multitude of signals and underlines the importance of the fas operon in the pathogenicity of R. fascians.

  4. Biosynthesis of auxin by the gram-positive phytopathogen Rhodococcus fascians is controlled by compounds specific to infected plant tissues.

    PubMed

    Vandeputte, Olivier; Oden, Sevgi; Mol, Adeline; Vereecke, Danny; Goethals, Koen; El Jaziri, Mondher; Prinsen, Els

    2005-03-01

    The role and metabolism of indole-3-acetic acid in gram-negative bacteria is well documented, but little is known about indole-3-acetic acid biosynthesis and regulation in gram-positive bacteria. The phytopathogen Rhodococcus fascians, a gram-positive organism, incites diverse developmental alterations, such as leafy galls, on a wide range of plants. Phenotypic analysis of a leafy gall suggests that auxin may play an important role in the development of the symptoms. We show here for the first time that R. fascians produces and secretes the auxin indole-3-acetic acid. Interestingly, whereas noninfected-tobacco extracts have no effect, indole-3-acetic acid synthesis is highly induced in the presence of infected-tobacco extracts when tryptophan is not limiting. Indole-3-acetic acid production by a plasmid-free strain shows that the biosynthetic genes are located on the bacterial chromosome, although plasmid-encoded genes contribute to the kinetics and regulation of indole-3-acetic acid biosynthesis. The indole-3-acetic acid intermediates present in bacterial cells and secreted into the growth media show that the main biosynthetic route used by R. fascians is the indole-3-pyruvic acid pathway with a possible rate-limiting role for indole-3-ethanol. The relationship between indole-3-acetic acid production and the symptoms induced by R. fascians is discussed.

  5. Rhodococcus fascians infection accelerates progression of tobacco BY-2 cells into mitosis through rapid changes in plant gene expression.

    PubMed

    Vandeputte, Olivier; Vereecke, Danny; Mol, Adeline; Lenjou, Marc; Van Bockstaele, Dirk; El Jaziri, Mondher; Baucher, Marie

    2007-01-01

    * To characterize plant cell cycle activation following Rhodococcus fascians infection, bacterial impact on cell cycle progression of tobacco BY-2 cells was investigated. * S-phase-synchronized BY-2 cells were cocultivated with R. fascians and cell cycle progression was monitored by measuring mitotic index, cell cycle gene expression and flow cytometry parameters. Cell cycle alteration was further investigated by cDNA-AFLP (amplified fragment length polymorphism). * It was shown that cell cycle progression of BY-2 cells was accelerated only upon infection with bacteria whose virulence gene expression was induced by a leafy gall extract. Thirty-eight BY-2 genes showed a differential expression within 6 h post-infection. Among these, seven were previously associated with specific plant cell cycle phases (in particular S and G2/M phases). Several genes also showed a differential expression during leafy gall formation. * R. fascians-infected BY-2 cells provide a simple model to identify plant genes related to leafy gall development. R. fascians can also be regarded as a useful biotic agent to alter cell cycle progression and, thereby, gain a better understanding of cell cycle regulation in plants.

  6. The plasmid-encoded chloramphenicol-resistance protein of Rhodococcus fascians is homologous to the transmembrane tetracycline efflux proteins.

    PubMed

    Desomer, J; Vereecke, D; Crespi, M; Van Montagu, M

    1992-08-01

    The nucleotide sequence of the chloramphenicol-resistance gene (cmr) of Rhodococcus fascians NCPPB 1675 (located on the conjugative plasmid pRF2) allowed the identification of two possible open reading frames (ORFs), of which ORF1 was consistent with the mutational analysis. Biochemical analysis of cmr revealed that it does not encode an antibiotic-modifying enzyme. The amino acid sequence of ORF1 predicted a hydrophobic protein, with 12 putative membrane-spanning domains, homologous to proteins involved in the efflux of tetracycline across the plasma membrane. Expression of the cmr gene was induced by addition of chloramphenicol to the growth media. The promoter of this gene was restricted to 50 bp upstream from a 200 bp 5'-untranslated mRNA region, the latter containing two inverted repeats. At the amino acid level, the cmr gene is 52% identical to a previously identified chloramphenicol-resistance determinant in Streptomyces lividans, indicating a wider dispersion of this type of cmr gene among the actinomycetes.

  7. High-cell-density batch fermentation of Rhodococcus opacus PD630 using a high glucose concentration for triacylglycerol production.

    PubMed

    Kurosawa, Kazuhiko; Boccazzi, Paolo; de Almeida, Naomi M; Sinskey, Anthony J

    2010-06-01

    Biodiesel, monoalkyl esters of long-chain fatty acids with short-chain alcohols derived from triacylglycerols (TAGs), can be produced from renewable biomass sources. Recently, there has been interest in producing microbial oils from oleaginous microorganisms. Rhodococcus opacus PD630 is known to accumulate large amounts of TAGs. Following on these earlier works we demonstrate that R. opacus PD630 has the uncommon capacity to grow in defined media supplemented with glucose at a concentration of 300 g l(-1) during batch-culture fermentations. We found that we could significantly increase concentrations of both glucose and (NH4)2SO4 in the production medium resulting in a dramatic increase in fatty acid production when pH was controlled. We describe the experimental design protocol used to achieve the culture conditions necessary to obtain both high-cell-density and TAG accumulation; specifically, we describe the importance of the C/N ratio of the medium composition. Our bioprocess results demonstrate that R. opacus PD630 grown in batch-culture with an optimal production medium containing 240 g l(-1) glucose and 13.45 g l(-1) (NH4)2SO4 (C/N of 17.8) yields 77.6 g l(-1) of cell dry weight composed of approximately 38% TAGs indicating that this strain holds great potential as a future source of industrial biodiesel on starchy cellulosic feedstocks that are glucose polymers.

  8. Phenol degrading ability of Rhodococcus pyrinidivorans and Pseudomonas aeruginosa isolated from activated sludge plants in South Africa.

    PubMed

    Kumari, Sheena; Chetty, Dereshen; Ramdhani, Nishani; Bux, Faizal

    2013-01-01

    Phenol, a common constituent in many industrial wastewaters is a major pollutant and has several adverse effects on the environment. The potential of various microorganisms to utilize phenol for their metabolic activity has been observed to be an effective means of remediating this toxic compound from the environment particularly wastewater. Five indigenous bacterial isolates (PD1-PD5) were obtained from phenol bearing industrial wastewater using the mineral salts medium. The isolates were further characterized based on their morphology, biochemical reactions and 16S rRNA phylogeny. The 16S rRNA sequence analysis using universal primers (27f/1492r) revealed that PD1, PD2, PD3 and PD4 were closely related to the actinomycete Rhodococcus pyrinidivorans (99%) and PD5 to Pseudomonas aeruginosa (99%). Growth kinetic patterns and phenol degradation abilities of the two representative isolates (PD1 and PD5) were also evaluated. Both the species were effective in utilizing phenol as the sole carbon source and could tolerate phenol concentrations of up to 500 to 600 mg/L. The ability of these isolates to utilize higher concentrations of phenol as their sole carbon source makes them potential candidates and better competitors in the bioremediation process.

  9. Interaction of a trehalose lipid biosurfactant produced by Rhodococcus erythropolis 51T7 with a secretory phospholipase A2.

    PubMed

    Zaragoza, Ana; Teruel, José A; Aranda, Francisco J; Ortiz, Antonio

    2013-10-15

    Trehalose-containing glycolipid biosurfactants form an emerging group of interesting compounds, which alter the structure and properties of phospholipid membranes, and interact with enzymatic and non-enzymatic proteins. Phospholipases A2 constitute a class of enzymes that hydrolyze the sn-2 ester of glycerophospholipids, and are classified into secreted phospholipases A2 (sPLA2) and intracellular phospholipases A2. In this work, pancreatic sPLA2 was chosen as a model enzyme to study the effect of the trehalose lipid biosurfactant on enzymes acting on interfaces. By using this enzyme, it is possible to study the modulation of enzyme activity, either by direct interaction of the biosurfactant with the protein, or as a result of the incorporation of the glycolipid on the phospholipid target membrane. It is shown that the succinoyl trehalose lipid isolated from Rhodococcus erythropolis 51T7 interacts with porcine pancreatic sPLA2 and inhibits its catalytic activity. Two modes of inhibition are observed, which are clearly differentiated by its timescale. First, a slow inhibition of sPLA2 activity upon preincubation of the enzyme with trehalose lipid in the absence of substrate is described. Second, incorporation of trehalose lipid into the phospholipid target membrane gives rise to a fast enzyme inhibition. These results are discussed in the light of previous data on sPLA2 inhibitors and extend the list of interesting biological activities reported for this R. erythropolis trehalose lipid biosurfactant.

  10. Isolation, characterization of Rhodococcus sp. P14 capable of degrading high-molecular-weight polycyclic aromatic hydrocarbons and aliphatic hydrocarbons.

    PubMed

    Song, Xiaohui; Xu, Yan; Li, Gangmin; Zhang, Ying; Huang, Tongwang; Hu, Zhong

    2011-10-01

    Rhodococcus sp. P14 was isolated from crude oil-contaminated sediments. This strain was capable of utilizing three to five rings polycyclic aromatic hydrocarbons (PAHs) including phenanthrene (Phe), pyrene (Pyr), and benzo[a]pyrene (BaP) as a sole carbon and energy source. After cultivated with 50mg/L of each PAH, strain P14 removed 43% Phe, 34% Pyr and 30% BaP in 30 d. Four different hydroxyphenanthrene products derived from Phe by strain P14 (1,2,3,4-hydroxyphenanthrene) were detected using SPME-GC-MS. Strain P14 also was capable of degrading mineral oil with n-alkanes of C17 to C21 carbon chain length. Compared with glucose-grown cells, PAHs-grown cells had decreased contents of shorter-chain length fatty acids (≤ C16:0), increased contents of C18:0, Me-C19:0 and disappeared odd-number carbon chain fatty acids. The contents of unsaturated C19:1, Me-C19:0 increased and C18:0 decreased in mineral oil-grown cells. At the same time, the strain P14 tended to float when cultivated in mineral oil-supplemented liquid medium. The degradation capability of P14 to alkane and PAHs and its floating characteristics will be very helpful for future's application in oil-spill bioremediation.

  11. Genetic and Biochemical Characterization of a Novel Monoterpene ɛ-Lactone Hydrolase from Rhodococcus erythropolis DCL14

    PubMed Central

    van der Vlugt-Bergmans, Cécile J. B; van der Werf, Mariët J.

    2001-01-01

    A monoterpene ɛ-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is active with (4R)-4-isopropenyl-7-methyl-2-oxo-oxepanone and (6R)-6-isopropenyl-3-methyl-2-oxo-oxepanone, lactones derived from (4R)-dihydrocarvone, and 7-isopropyl-4-methyl-2-oxo-oxepanone, the lactone derived from menthone. Both enantiomers of 4-, 5-, 6-, and 7-methyl-2-oxo-oxepanone were converted at equal rates, suggesting that the enzyme is not stereoselective. Maximal enzyme activity was measured at pH 9.5 and 30°C. Determination of the N-terminal amino acid sequence of purified MLH enabled cloning of the corresponding gene by a combination of PCR and colony screening. The gene, designated mlhB (monoterpene lactone hydrolysis), showed up to 43% similarity to members of the GDXG family of lipolytic enzymes. Sequencing of the adjacent regions revealed two other open reading frames, one encoding a protein with similarity to the short-chain dehydrogenase reductase family and the second encoding a protein with similarity to acyl coenzyme A dehydrogenases. Both enzymes are possibly also involved in the monoterpene degradation pathways of this microorganism. PMID:11157238

  12. A 3-D airway epithelial cell and macrophage co-culture system to study Rhodococcus equi infection.

    PubMed

    Schwab, Ute; Caldwell, Shannon; Matychak, Mary-Beth; Felippe, Julia

    2013-07-15

    We developed a 3-D equine bronchial epithelial cell (BEC) culture that fully differentiates into ciliary beating and mucus producing cells. Using this system, we evaluated how mucus affects the phagocytic activity of macrophages. Adult horse monocyte-derived macrophages were incubated with Rhodococcus equi for 4h either in the mucus layer of in vitro generated airway epithelium or on collagen coated membranes. Using light and electron microscopy, we noted that the number of macrophages with intracellular bacteria, and the number of intracellular bacteria per macrophage were lower in the presence of mucus. TNFα measurements revealed that the presence of BECs promoted TNFα production by R. equi-infected macrophages; a decrease in TLR-2 (involved in R. equi recognition) and an increase in EGF-R (involved in mucin production) mRNA expression were also noted. Interestingly, when foal macrophages were added to foal BECs, we made the opposite observation, i.e. many macrophages were loaded with R. equi. Our in vitro bronchial system shows great potential for the identification of mechanisms how BECs and mucus play a role in phagocyte activation and bacterial clearance. Further studies using this system will show whether the airway environment in the foal responds differently to R. equi infection.

  13. Improvement of phytoremediation of an aged petroleum hydrocarbon-contaminated soil by Rhodococcus erythropolis CD 106 strain.

    PubMed

    Płociniczak, Tomasz; Fic, Ewa; Pacwa-Płociniczak, Magdalena; Pawlik, Małgorzata; Piotrowska-Seget, Zofia

    2017-07-03

    The aim of this study was to assess the impact of soil inoculation with the Rhodococcus erythropolis CD 106 strain on the effectiveness of the phytoremediation of an aged hydrocarbon-contaminated [approx. 1% total petroleum hydrocarbon (TPH)] soil using ryegrass (Lolium perenne). The introduction of CD 106 into the soil significantly increased the biomass of ryegrass and the removal of hydrocarbons in planted soil. The fresh weight of the shoots and roots of plants inoculated with CD 106 increased by 49% and 30%, respectively. After 210 days of the experiment, the concentration of TPH was reduced by 31.2%, whereas in the planted, non-inoculated soil, it was reduced by 16.8%. By contrast, the concentration of petroleum hydrocarbon decreased by 18.7% in non-planted soil bioaugmented with the CD 106 strain. The rifampicin-resistant CD 106 strain survived after inoculation into soil and was detected in the soil during the entire experimental period, but the number of CD 106 cells decreased constantly during the enhanced phytoremediation and bioaugmentation experiments. The plant growth-promoting and hydrocarbon-degrading properties of CD 106, which are connected with its long-term survival and limited impact on autochthonous microflora, make this strain a good candidate for improving the phytoremediation efficiency of soil contaminated with hydrocarbons.

  14. Evaluation of a commercially available hyperimmune plasma product for prevention of naturally acquired pneumonia caused by Rhodococcus equi in foals.

    PubMed

    Giguère, Steeve; Gaskin, Jack M; Miller, Corey; Bowman, James L

    2002-01-01

    To determine efficacy of a commercially available hyperimmune plasma product for prevention of naturally acquired pneumonia caused by Rhodococcus equi in foals. Randomized clinical trial. 165 foals. Foals were randomly assigned to 1 of 2 groups (hyperimmune plasma or nontreated controls). Foals with failure of passive transfer (FPT) of immunity were treated with hyperimmune plasma and evaluated as a third group. Foals that received plasma were given 950 ml between 1 and 10 days of age and between 30 and 50 days of age. A tracheobronchial aspirate was obtained from foals with clinical signs of respiratory tract disease for bacteriologic culture. A significant difference in incidence of pneumonia caused by R equi in foals with adequate passive transfer was not detected between foals that received plasma (19.1%) and nontreated foals (30%). Of 13 foals without FPT that received plasma and developed pneumonia caused by R equi, 12 developed disease prior to administration of the second dose of hyperimmune plasma. Incidence of undifferentiated pneumonia of all causes was not different between groups. Intravenous administration of the commercially available hyperimmune plasma was safe, and the product contained high concentrations of anti-R equi antibodies. However, within this limited foal population, the difference in incidence of pneumonia caused by R equi observed between foals that received plasma and control foals was not significant.

  15. Identification of pathogens and virulence profile of Rhodococcus equi and Escherichia coli strains obtained from sand of parks

    PubMed Central

    Fernandes, M.C.; Takai, S.; Leite, D.S.; Pinto, J.P.A.N.; Brandão, P.E.; Santarém, V.A.; Listoni, F.J.P.; Da Silva, A.V.; Ribeiro, M.G.

    2013-01-01

    The identification of pathogens of viral (Rotavirus, Coronavirus), parasitic (Toxocara spp.) and bacterial (Escherichia coli, Salmonella spp., Rhodococcus equi) origin shed in feces, and the virulence profile of R. equi and E. coli isolates were investigated in 200 samples of sand obtained from 40 parks, located in central region of state of Sao Paulo, Brazil, using different diagnostic methods. From 200 samples analyzed, 23 (11.5%) strains of R. equi were isolated. None of the R. equi isolates showed a virulent (vapA gene) or intermediately virulent (vapB gene) profiles. Sixty-three (31.5%) strains of E. coli were identified. The following genes encoding virulence factors were identified in E. coli: eae, bfp, saa, iucD, papGI, sfa and hly. Phylogenetic classification showed that 63 E. coli isolates belonged to groups B1 (52.4%), A (25.4%) and B2 (22.2%). No E. coli serotype O157:H7 was identified. Eggs of Toxocara sp. were found in three parks and genetic material of bovine Coronavirus was identified in one sample of one park. No Salmonella spp. and Rotavirus isolates were identified in the samples of sand. The presence of R. equi, Toxocara sp, bovine Coronavirus and virulent E. coli isolates in the environment of parks indicates that the sanitary conditions of the sand should be improved in order to reduce the risks of fecal transmission of pathogens of zoonotic potential to humans in these places. PMID:24294244

  16. Evaluation of biosurfactants grown in corn oil by Rhodococcus rhodochrous on removing of heavy metal ion from aqueous solution

    NASA Astrophysics Data System (ADS)

    Suryanti, Venty; Hastuti, Sri; Pujiastuti, Dwi

    2016-02-01

    The potential application of biosurfactants to remove heavy metal ion from aqueous solution by batch technique was examined. The glycolipids type biosurfactants were grown in a media containing of 20% v/v corn oil with 7 days of fermentation by Rhodococcus rhodochrous. The biosurfactants reduced the surface tension of water of about 51% from 62 mN/m to 30 mN/m. The biosurfactant increased the E24 of water-palm oil emulsion of about 55% from 43% to 97% and could maintain this E24 value of above 50% for up to 9 days. Heavy metal ion removal, in this case cadmium ion, by crude and patially purified biosurfactants has been investigated from aqueous solution at pH 6. Adsorption capacity of Cd(II) ion by crude biosurfactant with 5 and 10 minutes of contact times were 1.74 and 1.82 mg/g, respectively. Additionally, the adsorption capacity of Cd(II) ion by partially purified biosurfactant with 5 and 10 minutes of contact times were 0.79 and 1.34 mg/g, respectively. The results demonstrated that the adsorption capacity of Cd(II) ion by crude biosurfactant was higher than that of by partially purified biosurfactant. The results suggested that the biosurfactant could be used in the removal of heavy metal ions from aqueous solution.

  17. Rhodococcus erythropolis cells adapt their fatty acid composition during biofilm formation on metallic and non-metallic surfaces.

    PubMed

    Rodrigues, Carlos J C; de Carvalho, Carla C C R

    2015-12-01

    Several parameters are involved in bacterial adhesion and biofilm formation including surface type, medium composition and cellular surface hydrophobicty. When the cells are placed inside tubes, parameters such as oxygen availability should also influence cell adhesion. To understand which cellular lipids are involved in the molecular events of biofilm formation in Rhodococcus erythropolis, cell adhesion was promoted on different metallic and non-metallic surfaces immersed in culture media. These cells were able to modulate the fatty acid composition of the cell membrane in response to both the surface to which they adhered and the growth medium used. To assess the response of the cells to both surfaces and operational conditions, biofilms were also promoted inside a reactor built with five different types of tubes and with medium recirculation. The biofilm biomass could be directly related not to the hydrophobicity of the tubes used but to the oxygen permeability of the tubes. Besides this, cell age influenced the adhesion of the R. erythropolis cells to the tubes. Principal component analysis showed that the lipid composition of the cells could separate cells attached to metallic from those on non-metallic surfaces in the plane formed by PC1 and PC2, and influence biofilm biomass.

  18. Optimization of biosurfactant production in soybean oil by rhodococcus rhodochrous and its utilization in remediation of cadmium-contaminated solution

    NASA Astrophysics Data System (ADS)

    Suryanti, Venty; Hastuti, Sri; Andriani, Dewi

    2016-02-01

    Biosurfactant production by Rhodococcus rhodochrous in soybean oil was developed, where the effect of medium composition and fermentation time were evaluated. The optimum condition for biosurfactant production was achieved when a medium containing 30 g/L TSB (tryptic soy broth) and 20% v/v soybean oil was used as media with 7 days of fermentation. Biosurfactant was identified as glycolipids type biosurfactant which had critical micelle concentration (CMC) value of 896 mg/L. The biosurfactant had oil in water emulsion type and was able to reduce the surface tension of palm oil about 52% which could stabilize the emulsion up to 12 days. The batch removal of cadmium metal ion by crude and partially purified biosurfactants have been examined from synthetic aqueous solution at pH 6. The results exhibited that the crude biosurfactant had a much better adsorption ability of Cd(II) than that of partially purified biosurfactant. However, it was found that there was no significant difference in the adsorption of Cd(II) with 5 and 10 minutes of contact time. The results indicated that the biosurfactant could be used in remediation of heavy metals from contaminated aqueous solution.

  19. Genomic and Functional Analyses of Rhodococcus equi Phages ReqiPepy6, ReqiPoco6, ReqiPine5, and ReqiDocB7 ▿

    PubMed Central

    Summer, E. J.; Liu, M.; Gill, J. J.; Grant, M.; Chan-Cortes, T. N.; Ferguson, L.; Janes, C.; Lange, K.; Bertoli, M.; Moore, C.; Orchard, R. C.; Cohen, N. D.; Young, R.

    2011-01-01

    The isolation and results of genomic and functional analyses of Rhodococcus equi phages ReqiPepy6, ReqiDocB7, ReqiPine5, and ReqiPoco6 (hereafter referred to as Pepy6, DocB7, Pine5, and Poco6, respectively) are reported. Two phages, Pepy6 and Poco6, more than 75% identical, exhibited genome organization and protein sequence likeness to Lactococcus lactis phage 1706 and clostridial prophage elements. An unusually high fraction, 27%, of Pepy6 and Poco6 proteins were predicted to possess at least one transmembrane domain, a value much higher than the average of 8.5% transmembrane domain-containing proteins determined from a data set of 36,324 phage protein entries. Genome organization and protein sequence comparisons place phage Pine5 as the first nonmycobacteriophage member of the large Rosebush cluster. DocB7, which had the broadest host range among the four isolates, was not closely related to any phage or prophage in the database, and only 23 of 105 predicted encoded proteins could be assigned a functional annotation. Because of the relationship of Rhodococcus to Mycobacterium, it was anticipated that these phages should exhibit some of the features characteristic of mycobacteriophages. Traits that were identified as shared by the Rhodococcus phages and mycobacteriophages include the prevalent long-tailed morphology and the presence of genes encoding LysB-like mycolate-hydrolyzing lysis proteins. Application of DocB7 lysates to soils amended with a host strain of R. equi reduced recoverable bacterial CFU, suggesting that phage may be useful in limiting R. equi load in the environment while foals are susceptible to infection. PMID:21097585

  20. [Cloning and expression of Micrococcus luteus IAM 14879 Rpf and its role in the recovery of the VBNC state in Rhodococcus sp. DS471].

    PubMed

    Ding, Linxian; Zhang, Pinghua; Hong, Huachang; Lin, Hongjun; Yokota, Akira

    2012-01-01

    The purpose of the present study was to produce the Rpf (resuscitation promoting factor) protein by cloning and expressing the rpf gene, secreted by Micrococcus luteus IAM 14879, in Escherichia coli and to evaluate its role in the recovery of the VBNC (viable but non-culturable) state in high-GC Gram-positive bacteria. Genomic DNA was extracted from Micrococcus luteus IAM 14879 and the rpf gene was amplified by PCR using specific primers. The PCR products was purified, cloned into a pET15b expression vector, and transformed into Escherichia coli BL21 (DE3). Then the pET15b plasmid expression vector was used to confirm the purification of the recombinant proteins via SDS-PAGE. The VBNC state cells from the high-GC Gram-positive bacteria, Rhodococcus sp. DS471, were used to confirm the promotion and recovery of growth capacity. Rhodococcus sp. DS471 were isolated from soil and closely related to Micrococcus luteus IAM 14879. The gene sequences confirmed that the rpf gene from Micrococcus luteus IAM 14879 that was expressed in Escherichia coli, was 672 bp. SDS-PAGE analysis showed that the recombinant Rpf protein was obtained successfully, and further studies showed it capable of promoting the recovery of the VBNC state by about 100-fold relative to the control. Rpf of Micrococus luteus IAM 14879 can be successfully cloned and expressed in Escherichia coli and shows a strong ability to promote the recovery of the VBNC state of cells of Rhodococcus sp. DS471.

  1. Permeabilization induced by lipid II-targeting lantibiotic nisin and its effect on the bioconversion of vitamin D3 to 25-hydroxyvitamin D3 by Rhodococcus erythropolis.

    PubMed

    Imoto, Noriko; Nishioka, Taiki; Tamura, Tomohiro

    2011-02-18

    Vitamin D3 (VD3) is a fat-soluble prohormone in mammals. VD3 is inert and must be activated by hydroxylation at the C-25 and C-1α positions to exert its biological activity. We recently accomplished the bioconversion of VD3 to 25(OH)VD3 with a recombinant strain of Rhodococcus erythropolis and found that the permeability of VD3 into the cytoplasm may be the rate-limiting step of 25(OH)VD3 production (Sallam et al., 2010). When the cells were treated with the lipid II-targeting lantibiotic nisin, the permeability of green chemiluminescent cyclodextrin (GCCD), which is used as a model substrate instead of VD3-partially methylated-β-cyclodextrin (PMCD) complex, was drastically induced. Nisin also induced VD3 hydroxylation, and the rate was correlated with the expression levels of Vdh and its redox partner proteins. In the bioconversion reaction, the stability of the redox partner proteins and the additional NADH-regenerating system are crucial for VD3 hydroxylation. The degradation rate of the [2Fe-2S] cluster of ferredoxin ThcC from R. erythropolis NI86/21 is faster than that of AciB from Acinetobacter sp. OC4. Therefore, the nisin-treated R. erythropolis cells coexpressing Vdh and AciBC (1176.5 μg) exhibited much greater 25(OH)VD3 production than the cells coexpressing Vdh and ThcCD (431.7 μg) after four consecutive 16 h reactions. These results suggest that nisin forms nisin-lipid II pore complexes in the Rhodococcus membrane that increase the accessibility of VD3-PMCD complexes to the inside of the cells. Furthermore, nisin-treated Rhodococcus cells can be utilized for the bioconversion of other fat-soluble chemicals. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Draft Genome Sequence of Rhodococcus rhodnii Strain LMG5362, a Symbiont of Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae), the Principle Vector of Trypanosoma cruzi

    PubMed Central

    Pachebat, Justin A.; van Keulen, Geertje; Whitten, Miranda M. A.; Girdwood, Susan; Del Sol, Ricardo; Dyson, Paul J.

    2013-01-01

    We report the 4,385,577-bp high-quality draft assembly of the bacterial symbiont Rhodococcus rhodnii strain LMG5362, isolated from the gut of Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae), the principle vector of the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. This sequence might provide useful information for subsequent studies of the symbiotic relationship between Rd. prolixus and Rc. rhodnii, while also providing a starting point for the development of biotechnological applications for the control of Rd. prolixus. PMID:23788540

  3. Immobilization of Rhodococcus erythropolis B4 on radiation crosslinked poly(vinylpyrrolidone) hydrogel: Application to the degradation of polycyclic aromatic hydrocarbons

    NASA Astrophysics Data System (ADS)

    Djefal-Kerrar, A.; Gais, S.; Ouallouche, K.; Nacer Khodja, A.; Mahlous, M.; Hacène, H.

    2007-12-01

    A poly(vinylpyrrolidone) (PVP) hydrogel crosslinked by gamma radiation was used to immobilize, by adsorption, Rhodococcus erythropolis B4 strain. Immobilized cells were tested for their capacity to degrade naphthalene and anthracene, under aerobic conditions. The results showed that, the strain fixed is capable of growing in the presence of naphthalene or anthracene as a unique source of carbon. It was also shown that, the fixed strain can be preserved by freeze-drying for further use. The biodegradation capacity was improved during the second use.

  4. Mg2+-Dependent Control of the Spatial Arrangement of Rhodococcus erythropolis PR4 Cells in Aqueous-Alkane Two Phase Culture Containing n-Dodecane

    PubMed Central

    Takihara, Hayato; Akase, Yumiko; Sunairi, Michio; Iwabuchi, Noriyuki

    2016-01-01

    We recently reported that a close relationship exists between alkane carbon-chain length, cell growth, and translocation frequency in Rhodococcus. In the present study, we examined the regulation of the spatial arrangement of cells in aqueous-alkane two phase cultures. An analysis of the effects of minerals on cell localization revealed that changes in the concentration of MgSO4 in two phase cultures containing n-dodecane (C12) altered cell localization from translocation to adhesion and vice versa. Our results indicate that the spatial arrangement of cells in two phase culture systems is controlled through the regulation of MgSO4 concentrations. PMID:27180641

  5. Portable exhausters POR-004 SKID B, POR-005 SKID C, POR-006 SKID D storage plan

    SciTech Connect

    Nelson, O.D.

    1997-09-04

    This document provides a storage plan for portable exhausters POR-004 SKID B, POR-005 SKID C, AND POR-006 SKID D. The exhausters will be stored until they are needed by the TWRS (Tank Waste Remediation Systems) Saltwell Pumping Program. The storage plan provides criteria for portable exhauster storage, periodic inspections during storage, and retrieval from storage.

  6. A New Modified ortho Cleavage Pathway of 3-Chlorocatechol Degradation by Rhodococcus opacus 1CP: Genetic and Biochemical Evidence

    PubMed Central

    Moiseeva, Olga V.; Solyanikova, Inna P.; Kaschabek, Stefan R.; Gröning, Janosch; Thiel, Monika; Golovleva, Ludmila A.; Schlömann, Michael

    2002-01-01

    The 4-chloro- and 2,4-dichlorophenol-degrading strain Rhodococcus opacus 1CP has previously been shown to acquire, during prolonged adaptation, the ability to mineralize 2-chlorophenol. In addition, homogeneous chlorocatechol 1,2-dioxygenase from 2-chlorophenol-grown biomass has shown relatively high activity towards 3-chlorocatechol. Based on sequences of the N terminus and tryptic peptides of this enzyme, degenerate PCR primers were now designed and used for cloning of the respective gene from genomic DNA of strain 1CP. A 9.5-kb fragment containing nine open reading frames was obtained on pROP1. Besides other genes, a gene cluster consisting of four chlorocatechol catabolic genes was identified. As judged by sequence similarity and correspondence of predicted N termini with those of purified enzymes, the open reading frames correspond to genes for a second chlorocatechol 1,2-dioxygenase (ClcA2), a second chloromuconate cycloisomerase (ClcB2), a second dienelactone hydrolase (ClcD2), and a muconolactone isomerase-related enzyme (ClcF). All enzymes of this new cluster are only distantly related to the known chlorocatechol enzymes and appear to represent new evolutionary lines of these activities. UV overlay spectra as well as high-pressure liquid chromatography analyses confirmed that 2-chloro-cis,cis-muconate is transformed by ClcB2 to 5-chloromuconolactone, which during turnover by ClcF gives cis-dienelactone as the sole product. cis-Dienelactone was further hydrolyzed by ClcD2 to maleylacetate. ClcF, despite its sequence similarity to muconolactone isomerases, no longer showed muconolactone-isomerizing activity and thus represents an enzyme dedicated to its new function as a 5-chloromuconolactone dehalogenase. Thus, during 3-chlorocatechol degradation by R. opacus 1CP, dechlorination is catalyzed by a muconolactone isomerase-related enzyme rather than by a specialized chloromuconate cycloisomerase. PMID:12218013

  7. Desulfurization activity and reusability of magnetite nanoparticle-coated Rhodococcus erythropolis FMF and R. erythropolis IGTS8 bacterial cells.

    PubMed

    Bardania, Hassan; Raheb, Jamshid; Mohammad-Beigi, Hossein; Rasekh, Behnam; Arpanaei, Ayyoob

    2013-01-01

    The application of Fe3 O4 nanoparticles to the separation of desulfurizing bacterial cells and their influence on the desulfurization activity and reusability of the two bacterial strains Rhodococcus erythropolis FMF and R. erythropolis IGTS8 were investigated. Magnetite nanoparticles were synthesized via the reverse coprecipitation method. Transmission electron microscopy (TEM) images showed that the magnetite nanoparticles had sizes of 5.35 ± 1.13 (F1 nanoparticles) and 8.74 ± 1.18 nm (F2 nanoparticles) when glycine was added during the synthesis of nanoparticles and when it was absent from the reaction mixture, respectively. Glycine was added after the synthesis of both F1 and F2 nanoparticles to stabilize the nanoparticle dispersion. TEM images of cells treated with magnetite nanoparticles indicated that F1 nanoparticles were immobilized on the surface of bacterial cells more evenly than the F2 nanoparticles. Desulfurization activities of the F1 magnetite nanoparticle-coated R. erythropolis FMF and R. erythropolis IGTS8 cells (with sulfur-removal percentage values of 70 ± 4 and 73 ± 3, respectively), as examined with the spectrophotometric Gibbs assay (based on dibenzothiophene degradation and sulfur-removal percentage), were not significantly different from those for the free bacterial cells (67 ± 3 and 69 ± 4, respectively). These results indicate that magnetite nanoparticles cannot affect the desulfurization activity of cells examined in this work. Isolation of bacterial cells from the suspension using a magnet and evaluation of desulfurization activity of separated cells showed that Fe3 O4 nanoparticles can provide a high-efficiency recovery of bacterial cells from a suspension, with the reused magnetite nanoparticle-coated bacterial cells being able to maintain their desulfurization activity efficiently. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

  8. Diverse effects of a biosurfactant from Rhodococcus ruber IEGM 231 on the adhesion of resting and growing bacteria to polystyrene.

    PubMed

    Kuyukina, Maria S; Ivshina, Irena B; Korshunova, Irina O; Stukova, Galina I; Krivoruchko, Anastasiya V

    2016-03-01

    This study evaluated the effects of a trehalolipid biosurfactant produced by Rhodococcus ruber IEGM 231 on the bacterial adhesion and biofilm formation on the surface of polystyrene microplates. The adhesion of Gram-positive (Arthrobacter simplex, Bacillus subtilis, Brevibacterium linens, Corynebacterium glutamicum, Micrococcus luteus) and Gram-negative (Escherichia coli, Pseudomonas fluorescencens) bacteria correlated differently with the cell hydrophobicity and surface charge. In particular, exponentially growing bacterial cells with increased hydrophobicities adhered stronger to polystyrene compared to more hydrophilic stationary phase cells. Also, a moderate correlation (0.56) was found between zeta potential and adhesion values of actively growing bacteria, suggesting that less negatively charged cells adhered stronger to polystyrene. Efficient biosurfactant concentrations (10-100 mg/L) were determined, which selectively inhibited (up to 76 %) the adhesion of tested bacterial cultures, however without inhibiting their growth. The biosurfactant was more active against growing bacteria rather than resting cells, thus showing high biofilm-preventing properties. Contact angle measurements revealed more hydrophilic surface of the biosurfactant-covered polystyrene compared to bare polystyrene, which allowed less adhesion of hydrophobic bacteria. Furthermore, surface free-energy calculations showed a decrease in the Wan der Waals (γ(LW)) component and an increase in the acid-based (γ(AB)) component caused by the biosurfactant coating of polysterene. However, our results suggested that the biosurfactant inhibited the adhesion of bacteria independently on their surface charges. AFM scanning revealed three-type biosurfactant structures (micelles, cord-like assemblies and large vesicles) formed on glass, depending on concentrations used, that could lead to diverse anti-adhesive effects against different bacterial species.

  9. Increasing awareness of Rhodococcus equi pulmonary infection in the immunocompetent adult: a rare infection with poor prognosis.

    PubMed

    Herath, Samantha; Lewis, Christopher; Nisbet, Mitzi

    2013-12-13

    The aim of this case report and review is to increase awareness of this uncommon infection with Rhodococcus equi (R. equi), in immunocompetent adults. R. equi is a soil-dwelling Gram-positive bacillus that frequently causes infection in grazing livestock. Human infection is rare and mostly limited to the immunocompromised hosts. We present a case of pneumonia caused by R. equi infection in a 55-year-old male builder who presented with cough, dyspnoea and night sweats, initially suspected to have pulmonary tuberculosis. Following biopsy of the mediastinal lymph nodes, R. equi was cultured, which is usually not a contaminant. Despite extensive investigations a host immune defect was not identified. The patient recovered after three months of combination antibiotic treatment, initially with intravenous vancomycin and meropenem followed by oral clarithromycin and rifampicin. To further clarify this rare disease we did a literature review that identified 26 adult patients with R. equi infection, without an identified host immunosuppressive condition. In this cohort, the median age at presentation was 53 years and infection holds a strong male predominance 19 (73%). An environmental exposure (e.g. farming, horse breeder) was found in 13 (50%). Ten (38%) of these patients had pulmonary infection. All deaths 3 (12%) occurred in the patients had pulmonary infection. R.equi is an infection that is difficult to diagnose and carries a high mortality if prompt treatment is not established. It is important to realise the potential for this disease to be misdiagnosed as pulmonary tuberculosis or community acquired pneumonia. Clinical suspicion is important especially if an environmental exposure is suspected.

  10. Age-related changes following in vitro stimulation with Rhodococcus equi of peripheral blood leukocytes from neonatal foals.

    PubMed

    Kachroo, Priyanka; Ivanov, Ivan; Seabury, Ashley G; Liu, Mei; Chowdhary, Bhanu P; Cohen, Noah D

    2013-01-01

    Rhodococcus equi is an intracellular bacterium primarily known as an equine pathogen that infects young foals causing a pyogranulomatuous pneumonia. The molecular mechanisms mediating the immune response of foals to R. equi are not fully elucidated. Hence, global genomic high-throughput tools like gene expression microarrays might identify age-related gene expression signatures and molecular pathways that contribute to the immune mechanisms underlying the inherent susceptibility of foals to disease caused by R. equi. The objectives of this study were 2-fold: 1) to compare the expression profiles at specific ages of blood leukocytes from foals stimulated with virulent R. equi with those of unstimulated leukocytes; and, 2) to characterize the age-related changes in the gene expression profile associated with blood leukocytes in response to stimulation with virulent R. equi. Peripheral blood leukocytes were obtained from 6 foals within 24 hours (h) of birth (day 1) and 2, 4, and 8 weeks after birth. The samples were split, such that half were stimulated with live virulent R. equi, and the other half served as unstimulated control. RNA was extracted and the generated cDNA was labeled with fluorescent dyes for microarray hybridizations using an equine microarray. Our findings suggest that there is age-related differential expression of genes involved in host immune response and immunity. We found induction of genes critical for host immunity against pathogens (MHC class II) only at the later time-points (compared to birth). While it appears that foals up to 8-weeks of age are able to initiate a protective inflammatory response against the bacteria, relatively decreased expression of various other immune-related genes points toward inherent diminished immune responses closer to birth. These genes and pathways may contribute to disease susceptibility in foals if infected early in life, and might thus be targeted for developing preventative or therapeutic strategies.

  11. Structural characterisation of the virulence-associated protein VapG from the horse pathogen Rhodococcus equi

    PubMed Central

    Okoko, Tebekeme; Blagova, Elena V.; Whittingham, Jean L.; Dover, Lynn G.; Wilkinson, Anthony J.

    2015-01-01

    Virulence and host range in Rhodococcus equi depends on the variable pathogenicity island of their virulence plasmids. Notable gene products are a family of small secreted virulence-associated proteins (Vaps) that are critical to intramacrophagic proliferation. Equine-adapted strains, which cause severe pyogranulomatous pneumonia in foals, produce a cell-associated VapA that is necessary for virulence, alongside five other secreted homologues. In the absence of biochemical insight, attention has turned to the structures of these proteins to develop a functional hypothesis. Recent studies have described crystal structures for VapD and a truncate of the VapA orthologue of porcine-adapted strains, VapB. Here, we crystallised the full-length VapG and determined its structure by molecular replacement. Electron density corresponding to the N-terminal domain was not visible suggesting that it is disordered. The protein core adopted a compact elliptical, anti-parallel β-barrel fold with β1–β2–β3–β8–β5–β6–β7–β4 topology decorated by a single peripheral α-helix unique to this family. The high glycine content of the protein allows close packing of secondary structural elements. Topologically, the surface has no indentations that indicate a nexus for molecular interactions. The distribution of polar and apolar groups on the surface of VapG is markedly uneven. One-third of the surface is dominated by exposed apolar side-chains, with no ionisable and only four polar side-chains exposed, giving rise to an expansive flat hydrophobic surface. Other surface regions are more polar, especially on or near the α-helix and a belt around the centre of the β-barrel. Possible functional significance of these recent structures is discussed. PMID:25746683

  12. Metabolism of 2-Chloro-4-Nitroaniline via Novel Aerobic Degradation Pathway by Rhodococcus sp. Strain MB-P1

    PubMed Central

    Khan, Fazlurrahman; Pal, Deepika; Vikram, Surendra; Cameotra, Swaranjit Singh

    2013-01-01

    2-chloro-4-nitroaniline (2-C-4-NA) is used as an intermediate in the manufacture of dyes, pharmaceuticals, corrosion inhibitor and also used in the synthesis of niclosamide, a molluscicide. It is marked as a black-listed substance due to its poor biodegradability. We report biodegradation of 2-C-4-NA and its pathway characterization by Rhodococcus sp. strain MB-P1 under aerobic conditions. The strain MB-P1 utilizes 2-C-4-NA as the sole carbon, nitrogen, and energy source. In the growth medium, the degradation of 2-C-4-NA occurs with the release of nitrite ions, chloride ions, and ammonia. During the resting cell studies, the 2-C-4-NA-induced cells of strain MB-P1 transformed 2-C-4-NA stoichiometrically to 4-amino-3-chlorophenol (4-A-3-CP), which subsequently gets transformed to 6-chlorohydroxyquinol (6-CHQ) metabolite. Enzyme assays by cell-free lysates prepared from 2-C-4-NA-induced MB-P1 cells, demonstrated that the first enzyme in the 2-C-4-NA degradation pathway is a flavin-dependent monooxygenase that catalyzes the stoichiometric removal of nitro group and production of 4-A-3-CP. Oxygen uptake studies on 4-A-3-CP and related anilines by 2-C-4-NA-induced MB-P1 cells demonstrated the involvement of aniline dioxygenase in the second step of 2-C-4-NA degradation. This is the first report showing 2-C-4-NA degradation and elucidation of corresponding metabolic pathway by an aerobic bacterium. PMID:23614030

  13. Rhodococcus equi’s Extreme Resistance to Hydrogen Peroxide Is Mainly Conferred by One of Its Four Catalase Genes

    PubMed Central

    Barbey, Corinne; Appourchaux, Anne-Cécile; Torelli, Riccardo; Sanguinetti, Maurizio; Laugier, Claire; Petry, Sandrine

    2012-01-01

    Rhodococcus equi is one of the most widespread causes of disease in foals aged from 1 to 6 months. R. equi possesses antioxidant defense mechanisms to protect it from reactive oxygen metabolites such as hydrogen peroxide (H2O2) generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxify hydrogen peroxide. Recently, an analysis of the R. equi 103 genome sequence revealed the presence of four potential catalase genes. We first constructed ΔkatA-, ΔkatB-, ΔkatC-and ΔkatD-deficient mutants to study the ability of R. equi to survive exposure to H2O2 in vitro and within mouse peritoneal macrophages. Results showed that ΔkatA and, to a lesser extent ΔkatC, were affected by 80 mM H2O2. Moreover, katA deletion seems to significantly affect the ability of R. equi to survive within murine macrophages. We finally investigated the expression of the four catalases in response to H2O2 assays with a real time PCR technique. Results showed that katA is overexpressed 367.9 times (±122.6) in response to exposure to 50 mM of H2O2 added in the stationary phase, and 3.11 times (±0.59) when treatment was administered in the exponential phase. In untreated bacteria, katB, katC and katD were overexpressed from 4.3 to 17.5 times in the stationary compared to the exponential phase. Taken together, our results show that KatA is the major catalase involved in the extreme H2O2 resistance capability of R. equi. PMID:22879963

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

    PubMed

    van der Werf, M J; van der Ven, C; Barbirato, F; Eppink, M H; de Bont, J A; van Berkel, W J

    1999-09-10

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

  15. Transcriptome of the quorum-sensing signal-degrading Rhodococcus erythropolis responds differentially to virulent and avirulent Pectobacterium atrosepticum.

    PubMed

    Kwasiborski, A; Mondy, S; Chong, T-M; Barbey, C; Chan, K-G; Beury-Cirou, A; Latour, X; Faure, D

    2015-05-01

    Social bacteria use chemical communication to coordinate and synchronize gene expression via the quorum-sensing (QS) regulatory pathway. In Pectobacterium, a causative agent of the blackleg and soft-rot diseases on potato plants and tubers, expression of the virulence factors is collectively controlled by the QS-signals N-acylhomoserine lactones (NAHLs). Several soil bacteria, such as the actinobacterium Rhodococcus erythropolis, are able to degrade NAHLs, hence quench the chemical communication and virulence of Pectobacterium. Here, next-generation sequencing was used to investigate structural and functional genomics of the NAHL-degrading R. erythropolis strain R138. The R. erythropolis R138 genome (6.7 Mbp) contained a single circular chromosome, one linear (250 kbp) and one circular (84 kbp) plasmid. Growth of R. erythropolis and P. atrosepticum was not altered in mixed-cultures as compared with monocultures on potato tuber slices. HiSeq-transcriptomics revealed that no R. erythropolis genes were differentially expressed when R. erythropolis was cultivated in the presence vs absence of the avirulent P. atrosepticum mutant expI, which is defective for QS-signal synthesis. By contrast 50 genes (<1% of the R. erythropolis genome) were differentially expressed when R. erythropolis was cultivated in the presence vs absence of the NAHL-producing virulent P. atrosepticum. Among them, quantitative real-time reverse-transcriptase-PCR confirmed that the expression of some alkyl-sulfatase genes decreased in the presence of a virulent P. atrosepticum, as well as deprivation of organic sulfur such as methionine, which is a key precursor in the synthesis of NAHL by P. atrosepticum.

  16. Two independently regulated cytochromes P-450 in a Rhodococcus rhodochrous strain that degrades 2-ethoxyphenol and 4-methoxybenzoate.

    PubMed Central

    Karlson, U; Dwyer, D F; Hooper, S W; Moore, E R; Timmis, K N; Eltis, L D

    1993-01-01

    A red-pigmented coryneform bacterium, identified as Rhodococcus rhodochrous strain 116, that grew on 2-ethoxyphenol and 4-methoxybenzoate as sole carbon and energy sources was isolated. Phylogenetic analysis based on the 16S rDNA sequences indicates that the strain clusters more closely to other rhodococci than to other gram-positive organisms with a high G + C content. Each of the abovementioned growth substrates was shown to induce a distinct cytochrome P-450: cytochrome P-450RR1 was induced by 2-ethoxyphenol, and cytochrome P-450RR2 was induced by 4-methoxybenzoate. A type I difference spectrum typical of substrate binding was induced in cytochrome P-450RR1 by both 2-ethoxyphenol (KS = 4.2 +/- 0.3 microM) and 2-methoxyphenol (KS = 2.0 +/- 0.1 microM), but not 4-methoxybenzoate or 4-ethoxybenzoate. Similarly, a type I difference spectrum was induced in cytochrome P-450RR2 by both 4-methoxybenzoate (KS = 2.1 +/- 0.1 microM) and 4-ethoxybenzoate (KS = 1.6 +/- 0.1 microM), but not 2-methoxyphenol or 2-ethoxyphenol. A purified polyclonal antiserum prepared against cytochrome P-450RR1 did not cross-react with cytochrome P-450RR2, indicating that the proteins are immunologically distinct. The cytochromes appear to catalyze the O-dealkylation of their respective substrates. The respective products of the O-dealkylation are further metabolized via ortho cleavage enzymes, whose expression is also regulated by the respective aromatic ethers. Images PMID:8444808

  17. Biodegradation of 4-nitrotoluene with biosurfactant production by Rhodococcus pyridinivorans NT2: metabolic pathway, cell surface properties and toxicological characterization.

    PubMed

    Kundu, Debasree; Hazra, Chinmay; Dandi, Navin; Chaudhari, Ambalal

    2013-11-01

    A novel 4-nitrotoluene-degrading bacterial strain was isolated from pesticides contaminated effluent-sediment and identified as Rhodococcus pyridinivorans NT2 based on morphological and biochemical properties and 16S rDNA sequencing. The strain NT2 degraded 4-NT (400 mg l(-1)) with rapid growth at the end of 120 h, reduced surface tension of the media from 71 to 29 mN m(-1) and produced glycolipidic biosurfactants (45 mg l(-1)). The biosurfactant was purified and characterized as trehalose lipids. The biosurfactant was stable in high salinity (10 % w/v NaCl), elevated temperatures (120 °C for 15 min) and a wide pH range (2.0-10.0). The noticeable changes during biodegradation were decreased hydrophobicity; an increase in degree of fatty acid saturation, saturated/unsaturated ratio and cyclopropane fatty acid. Biodegradation of 4-NT was accompanied by the accumulation of ammonium (NH4 (+)) and negligible amount of nitrite ion (NO2 (-)). Product stoichiometry showed a carbon (C) and nitrogen (N) mass balance of 37 and 35 %, respectively. Biodegradation of 4-NT proceeded by oxidation at the methyl group to form 4-nitrobenzoate, followed by reduction and hydrolytic deamination yielding protocatechuate, which was metabolized through β-ketoadipate pathway. In vitro and in vivo acute toxicity assays in adult rat (Rattus norvegicus) showed sequential detoxification and the order of toxicity was 4-NT >4-nitrobenzyl alcohol >4-nitrobenzaldehyde >4-nitrobenzoate > protocatechuate. Taken together, the strain NT2 could be used as a potential bioaugmentation candidate for the bioremediation of contaminated sites.

  18. Transcriptome of the quorum-sensing signal-degrading Rhodococcus erythropolis responds differentially to virulent and avirulent Pectobacterium atrosepticum

    PubMed Central

    Kwasiborski, A; Mondy, S; Chong, T-M; Barbey, C; Chan, K-G; Beury-Cirou, A; Latour, X; Faure, D

    2015-01-01

    Social bacteria use chemical communication to coordinate and synchronize gene expression via the quorum-sensing (QS) regulatory pathway. In Pectobacterium, a causative agent of the blackleg and soft-rot diseases on potato plants and tubers, expression of the virulence factors is collectively controlled by the QS-signals N-acylhomoserine lactones (NAHLs). Several soil bacteria, such as the actinobacterium Rhodococcus erythropolis, are able to degrade NAHLs, hence quench the chemical communication and virulence of Pectobacterium. Here, next-generation sequencing was used to investigate structural and functional genomics of the NAHL-degrading R. erythropolis strain R138. The R. erythropolis R138 genome (6.7 Mbp) contained a single circular chromosome, one linear (250 kbp) and one circular (84 kbp) plasmid. Growth of R. erythropolis and P. atrosepticum was not altered in mixed-cultures as compared with monocultures on potato tuber slices. HiSeq-transcriptomics revealed that no R. erythropolis genes were differentially expressed when R. erythropolis was cultivated in the presence vs absence of the avirulent P. atrosepticum mutant expI, which is defective for QS-signal synthesis. By contrast 50 genes (<1% of the R. erythropolis genome) were differentially expressed when R. erythropolis was cultivated in the presence vs absence of the NAHL-producing virulent P. atrosepticum. Among them, quantitative real-time reverse-transcriptase–PCR confirmed that the expression of some alkyl-sulfatase genes decreased in the presence of a virulent P. atrosepticum, as well as deprivation of organic sulfur such as methionine, which is a key precursor in the synthesis of NAHL by P. atrosepticum. PMID:25585922

  19. Characterization and application of bioflocculant prepared by Rhodococcus erythropolis using sludge and livestock wastewater as cheap culture media.

    PubMed

    Peng, Lanyan; Yang, Chunping; Zeng, Guangming; Wang, Lu; Dai, Chuanhua; Long, Zhiyong; Liu, Hongyu; Zhong, Yuanyuan

    2014-08-01

    A new bioflocculant was produced by culturing Rhodococcus erythropolis in a cheap medium. When culture pH was 7.0, inoculum size was 2 % (v/v), Na2HPO4 concentration was 0.5 g L(-1), and the ratio of sludge/livestock wastewater was 7:1 (v/v), a maximum flocculating rate of 87.6 % could be achieved. Among 13 different kinds of pretreatments for sludge, the optimal one was the thermal-alkaline pretreatment. Different from a bioflocculant produced in a standard medium, this bioflocculant was effective over a wide pH range from 2 to 12 with flocculating rates higher than 98 %. Approximately, 1.6 g L(-1) of crude bioflocculant could be harvested using cold ethanol for extraction. This bioflocculant showed color removal rates up to 80 % when applied to direct and disperse dye solutions, but only 23.0 % for reactive dye solutions. Infrared spectrum showed that the bioflocculant contained functional groups such as -OH, -NH2, and -CONH2. Components in the bioflocculant consisted of 91.2 % of polysaccharides, 7.6 % of proteins, and 1.2 % of DNA. When the bioflocculant and copper sulfate (CuSO4) were used together for decolorization in actual dye wastewater, the optimum decolorization conditions were specified by the response surface methodology as pH 11, bioflocculant dosage of 40 mg/L, and CuSO4 80 mg/L, under which a decolorization rate of 93.9 % could be reached.

  20. Physiological Adaptations Involved in Alkane Assimilation at a Low Temperature by Rhodococcus sp. Strain Q15†

    PubMed Central

    Whyte, L. G.; Slagman, S. J.; Pietrantonio, F.; Bourbonnière, L.; Koval, S. F.; Lawrence, J. R.; Inniss, W. E.; Greer, C. W.

    1999-01-01

    We examined physiological adaptations which allow the psychrotroph Rhodococcus sp. strain Q15 to assimilate alkanes at a low temperature (alkanes are contaminants which are generally insoluble and/or solid at low temperatures). During growth at 5°C on hexadecane or diesel fuel, strain Q15 produced a cell surface-associated biosurfactant(s) and, compared to glucose-acetate-grown cells, exhibited increased cell surface hydrophobicity. A transmission electron microscopy examination of strain Q15 grown at 5°C revealed the presence of intracellular electron-transparent inclusions and flocs of cells connected by an extracellular polymeric substance (EPS) when cells were grown on a hydrocarbon and morphological differences between the EPS of glucose-acetate-grown and diesel fuel-grown cells. A lectin binding analysis performed by using confocal scanning laser microscopy (CSLM) showed that the EPS contained a complex mixture of glycoconjugates, depending on both the growth temperature and the carbon source. Two glycoconjugates [β-d-Gal-(1-3)-d-GlcNAc and α-l-fucose] were detected only on the surfaces of cells grown on diesel fuel at 5°C. Using scanning electron microscopy, we observed strain Q15 cells on the surfaces of octacosane crystals, and using CSLM, we observed strain Q15 cells covering the surfaces of diesel fuel microdroplets; these findings indicate that this organism assimilates both solid and liquid alkane substrates at a low temperature by adhering to the alkane phase. Membrane fatty acid analysis demonstrated that strain Q15 adapted to growth at a low temperature by decreasing the degree of saturation of membrane lipid fatty acids, but it did so to a lesser extent when it was grown on hydrocarbons at 5°C; these findings suggest that strain Q15 modulates membrane fluidity in response to the counteracting influences of low temperature and hydrocarbon toxicity. PMID:10388690

  1. Characterization of a (2R,3R)-2,3-Butanediol Dehydrogenase from Rhodococcus erythropolis WZ010.

    PubMed

    Yu, Meilan; Huang, Meijuan; Song, Qingqing; Shao, Jianzhong; Ying, Xiangxian

    2015-04-20

    The gene encoding a (2R,3R)-2,3-butanediol dehydrogenase from Rhodococcus erythropolis WZ010 (ReBDH) was over-expressed in Escherichia coli and the resulting recombinant ReBDH was successfully purified by Ni-affinity chromatography. The purified ReBDH in the native form was found to exist as a monomer with a calculated subunit size of 37180, belonging to the family of the zinc-containing alcohol dehydrogenases. The enzyme was NAD(H)-specific and its optimal activity for acetoin reduction was observed at pH 6.5 and 55 °C. The optimal pH and temperature for 2,3-butanediol oxidation were pH 10 and 45 °C, respectively. The enzyme activity was inhibited by ethylenediaminetetraacetic acid (EDTA) or metal ions Al3+, Zn2+, Fe2+, Cu2+ and Ag+, while the addition of 10% (v/v) dimethyl sulfoxide (DMSO) in the reaction mixture increased the activity by 161.2%. Kinetic parameters of the enzyme showed lower Km values and higher catalytic efficiency for diacetyl and NADH in comparison to those for (2R,3R)-2,3-butanediol and NAD+. The activity of acetoin reduction was 7.7 times higher than that of (2R,3R)-2,3-butanediol oxidation when ReBDH was assayed at pH 7.0, suggesting that ReBDH-catalyzed reaction in vivo might favor (2R,3R)-2,3-butanediol formation rather than (2R,3R)-2,3-butanediol oxidation. The enzyme displayed absolute stereospecificity in the reduction of diacetyl to (2R,3R)-2,3-butanediol via (R)-acetoin, demonstrating its potential application on the synthesis of (R)-chiral alcohols.

  2. Potent Antiproliferative Cembrenoids Accumulate in Tobacco upon Infection with Rhodococcus fascians and Trigger Unusual Microtubule Dynamics in Human Glioblastoma Cells

    PubMed Central

    Nacoulma, Aminata P.; Megalizzi, Veronique; Pottier, Laurent R.; De Lorenzi, Manuela; Thoret, Sylviane; Dubois, Joëlle; Vandeputte, Olivier M.; Duez, Pierre; Vereecke, Danny; Jaziri, Mondher El

    2013-01-01

    Aims Though plant metabolic changes are known to occur during interactions with bacteria, these were rarely challenged for pharmacologically active compounds suitable for further drug development. Here, the occurrence of specific chemicals with antiproliferative activity against human cancer cell lines was evidenced in hyperplasia (leafy galls) induced when plants interact with particular phytopathogens, such as the Actinomycete Rhodococcus fascians. Methods We examined leafy galls fraction F3.1.1 on cell proliferation, cell division and cytoskeletal disorganization of human cancer cell lines using time-lapse videomicroscopy imaging, combined with flow cytometry and immunofluorescence analysis. We determined the F3.1.1-fraction composition by gas chromatography coupled to mass spectrometry. Results The leafy galls induced on tobacco by R. fascians yielded fraction F3.1.1 which inhibited proliferation of glioblastoma U373 cells with an IC50 of 4.5 µg/mL, F.3.1.1 was shown to increase cell division duration, cause nuclear morphological deformations and cell enlargement, and, at higher concentrations, karyokinesis defects leading to polyploidization and apoptosis. F3.1.1 consisted of a mixture of isomers belonging to the cembrenoids. The cellular defects induced by F3.1.1 were caused by a peculiar cytoskeletal disorganization, with the occurrence of fragmented tubulin and strongly organized microtubule aggregates within the same cell. Colchicine, paclitaxel, and cembrene also affected U373 cell proliferation and karyokinesis, but the induced microtubule rearrangement was very different from that provoked by F3.1.1. Altogether our data indicate that the cembrenoid isomers in F3.1.1 have a unique mode of action and are able to simultaneously modulate microtubule polymerization and stability. PMID:24167576

  3. Distribution of a Nocardia brasiliensis Catalase Gene Fragment in Members of the Genera Nocardia, Gordona, and Rhodococcus

    PubMed Central

    Vera-Cabrera, Lucio; Johnson, Wendy M.; Welsh, Oliverio; Resendiz-Uresti, Francisco L.; Salinas-Carmona, Mario C.

    1999-01-01

    An immunodominant protein from Nocardia brasiliensis, P61, was subjected to amino-terminal and internal sequence analysis. Three sequences of 22, 17, and 38 residues, respectively, were obtained and compared with the protein database from GenBank by using the BLAST system. The sequences showed homology to some eukaryotic catalases and to a bromoperoxidase-catalase from Streptomyces violaceus. Its identity as a catalase was confirmed by analysis of its enzymatic activity on H2O2 and by a double-staining method on a nondenaturing polyacrylamide gel with 3,3′-diaminobenzidine and ferricyanide; the result showed only catalase activity, but no peroxidase. By using one of the internal amino acid sequences and a consensus catalase motif (VGNNTP), we were able to design a PCR assay that generated a 500-bp PCR product. The amplicon was analyzed, and the nucleotide sequence was compared to the GenBank database with the observation of high homology to other bacterial and eukaryotic catalases. A PCR assay based on this target sequence was performed with primers NB10 and NB11 to confirm the presence of the NB10-NB11 gene fragment in several N. brasiliensis strains isolated from mycetoma. The same assay was used to determine whether there were homologous sequences in several type strains from the genera Nocardia, Rhodococcus, Gordona, and Streptomyces. All of the N. brasiliensis strains presented a positive result but only some of the actinomycetes species tested were positive in the PCR assay. In order to confirm these findings, genomic DNA was subjected to Southern blot analysis. A 1.7-kbp band was observed in the N. brasiliensis strains, and bands of different molecular weight were observed in cross-reacting actinomycetes. Sequence analysis of the amplicons of selected actinomycetes showed high homology in this catalase fragment, thus demonstrating that this protein is highly conserved in this group of bacteria. PMID:10325357

  4. The atf2 gene is involved in triacylglycerol biosynthesis and accumulation in the oleaginous Rhodococcus opacus PD630.

    PubMed

    Hernández, Martín A; Arabolaza, Ana; Rodríguez, Eduardo; Gramajo, Hugo; Alvarez, Héctor M

    2013-03-01

    Rhodococcus opacus PD630 is an oleaginous bacterium able to accumulate large amounts of triacylglycerols (TAG) in different carbon sources. The last reaction for TAG biosynthesis is catalyzed by the bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) enzymes encoded by atf genes. R. opacus PD630 possesses at least 17 putative atf homologous genes in its genome, but only atf1 and atf2 exhibited a significant DGAT activity when expressed in E. coli, as revealed in a previous study. The contribution of atf1 gene to TAG accumulation by strain PD630 has been demonstrated previously, although additional Atfs may also contribute to lipid accumulation, since the atf1-disrupted mutant is still able to produce significant amounts of TAG (Alvarez et al., Microbiology 154:2327-2335, 2008). In this study, we investigated the in vivo role of atf2 gene in TAG accumulation by R. opacus PD630 by using different genetic strategies. The atf2-disrupted mutant exhibited a decrease in TAG accumulation (up to 25-30 %, w/w) and an approximately tenfold increase in glycogen formation in comparison with the wild-type strain. Surprisingly, in contrast to single mutants, a double mutant generated by the disruption of atf1 and atf2 genes only showed a very low effect in TAG and in glycogen accumulation under lipid storage conditions. Overexpression of atf1 and atf2 genes in strain PD630 promoted an increase of approximately 10 % (w/w) in TAG accumulation, while heterologous expression of atf2 gene in Mycobacterium smegmatis caused an increase in TAG accumulation during cultivation in nitrogen-rich media. This study demonstrated that, in addition to atf1 gene, atf2 is actively involved in TAG accumulation by the oleaginous R. opacus PD630.

  5. Molecular characterization of Rhodococcus equi from horse-breeding farms by means of multiplex PCR for the vap gene family.

    PubMed

    Monego, Fernanda; Maboni, Franciele; Krewer, Cristina; Vargas, Agueda; Costa, Mateus; Loreto, Elgion

    2009-04-01

    This study evaluated the molecular characteristics of Rhodococcus equi isolates obtained from horses by a multiplex PCR assay that amplifies the vap gene family (vapA, -B, -C, -D, -E, -F, -G, and -H). A total of 180 R. equi isolates were studied from four different sources, namely healthy horse feces (112), soil (12), stalls (23), and clinical isolates (33) from horse-breeding farms. The technique was performed and confirmed by sequencing of amplified vap gene family controls. Thirty-two (17.8%) of the R. equi isolates were positive for the vapA gene and carried at least three other vap genes. All 147 isolates from equine feces, stalls, and soil failed to demonstrate any genes associated with virulence-inducing proteins. About 32 (97.0%) out of the 33 clinical equine isolates tested positive for the multiplex PCR assay for the vap gene family. They demonstrated six molecular profiles: 100% featured the vapA, vapD, and vapG genes, 86.6% vapF, 76.6% vapH, 43.3% vapC, 36.6% vapE, and none vapB. The most frequent molecular profile was vap A, -D, -F, G, and -H, where this profile was present in 37.5% of the strains. Moreover, there was no molecular epidemiological pattern for R. equi isolates that uniquely mapped to each horse-breeding farm studied. Our proposed technique allows the identification of eight members of the vap gene family (vapA, B, -C, -D, -E, -F, -G, and -H). It is a practical and efficient method of conducting clinical and epidemiological studies on R. equi isolates.

  6. Effects of age and macrophage lineage on intracellular survival and cytokine induction after infection with Rhodococcus equi.

    PubMed

    Berghaus, Londa J; Giguère, Steeve; Sturgill, Tracy L

    2014-07-15

    Rhodococcus equi, a facultative intracellular pathogen of macrophages, causes life-threatening pneumonia in foals and in people with underlying immune deficiencies. As a basis for this study, we hypothesized that macrophage lineage and age would affect intracellular survival of R. equi and cytokine induction after infection. Monocyte-derived and bronchoalveolar macrophages from 10 adult horses and from 10 foals (sampled at 1-3 days, 2 weeks, 1 month, 3 months, and 5 months of age) were infected ex vivo with virulent R. equi. Intracellular R. equi were quantified and mRNA expression of IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12 p40, IL-18, IFN-γ, and TNF-α was measured. Intracellular replication of R. equi was significantly (P<0.001) greater in bronchoalveolar than in monocyte-derived macrophages, regardless of age. Regardless of the macrophage lineage, replication of R. equi was significantly (P=0.002) higher in 3-month-old foals than in 3-day old foals, 2-week-old foals, 1-month-old foals, and adult horses. Expression of IL-4 mRNA was significantly higher in monocyte-derived macrophages whereas expression of IL-6, IL-18, and TNF-α was significantly higher in bronchoalveolar macrophages. Induction of IL-1β, IL-10, IL-12 p40, and IL-8 mRNA in bronchoalveolar macrophages of 1-3-day old foals was significantly higher than in older foals or adult horses. Preferential intracellular survival of R. equi in bronchoalveolar macrophages of juvenile horses may play a role in the pulmonary tropism of the pathogen and in the window of age susceptibility to infection.

  7. Identification and mutagenesis by allelic exchange of choE, encoding a cholesterol oxidase from the intracellular pathogen Rhodococcus equi.

    PubMed

    Navas, J; González-Zorn, B; Ladrón, N; Garrido, P; Vázquez-Boland, J A

    2001-08-01

    The virulence mechanisms of the facultative intracellular parasite Rhodococcus equi remain largely unknown. Among the candidate virulence factors of this pathogenic actinomycete is a secreted cholesterol oxidase, a putative membrane-damaging toxin. We identified and characterized the gene encoding this enzyme, the choE monocistron. Its protein product, ChoE, is homologous to other secreted cholesterol oxidases identified in Brevibacterium sterolicum and Streptomyces spp. ChoE also exhibits significant similarities to putative cholesterol oxidases encoded by Mycobacterium tuberculosis and Mycobacterium leprae. Genetic tools for use with R. equi are poorly developed. Here we describe the first targeted mutagenesis system available for this bacterium. It is based on a suicide plasmid, a selectable marker (the aacC4 apramycin resistance gene from Salmonella), and homologous recombination. The choE allele was disrupted by insertion of the aacC4 gene, cloned in pUC19 and introduced by electroporation in R. equi. choE recombinants were isolated at frequencies between 10(-2) and 10(-3). Twelve percent of the recombinants were double-crossover choE mutants. The choE mutation was associated with loss of cooperative (CAMP-like) hemolysis with sphingomyelinase-producing bacteria (Listeria ivanovii). Functional complementation was achieved by expression of choE from pVK173-T, a pAL5000 derivative conferring hygromycin resistance. Our data demonstrate that ChoE is an important cytolytic factor for R. equi. The highly efficient targeted mutagenesis procedure that we used to generate choE isogenic mutants will be a valuable tool for the molecular analysis of R. equi virulence.

  8. Structural characterisation of the virulence-associated protein VapG from the horse pathogen Rhodococcus equi.

    PubMed

    Okoko, Tebekeme; Blagova, Elena V; Whittingham, Jean L; Dover, Lynn G; Wilkinson, Anthony J

    2015-08-31

    Virulence and host range in Rhodococcus equi depends on the variable pathogenicity island of their virulence plasmids. Notable gene products are a family of small secreted virulence-associated proteins (Vaps) that are critical to intramacrophagic proliferation. Equine-adapted strains, which cause severe pyogranulomatous pneumonia in foals, produce a cell-associated VapA that is necessary for virulence, alongside five other secreted homologues. In the absence of biochemical insight, attention has turned to the structures of these proteins to develop a functional hypothesis. Recent studies have described crystal structures for VapD and a truncate of the VapA orthologue of porcine-adapted strains, VapB. Here, we crystallised the full-length VapG and determined its structure by molecular replacement. Electron density corresponding to the N-terminal domain was not visible suggesting that it is disordered. The protein core adopted a compact elliptical, anti-parallel β-barrel fold with β1-β2-β3-β8-β5-β6-β7-β4 topology decorated by a single peripheral α-helix unique to this family. The high glycine content of the protein allows close packing of secondary structural elements. Topologically, the surface has no indentations that indicate a nexus for molecular interactions. The distribution of polar and apolar groups on the surface of VapG is markedly uneven. One-third of the surface is dominated by exposed apolar side-chains, with no ionisable and only four polar side-chains exposed, giving rise to an expansive flat hydrophobic surface. Other surface regions are more polar, especially on or near the α-helix and a belt around the centre of the β-barrel. Possible functional significance of these recent structures is discussed.

  9. Localization of Low Copy Number Plasmid pRC4 in Replicating Rod and Non-Replicating Cocci Cells of Rhodococcus erythropolis PR4

    PubMed Central

    Singhi, Divya; Jain, Aayushi; Srivastava, Preeti

    2016-01-01

    Rhodococcus are gram-positive bacteria, which can exist in two different shapes rod and cocci. A number of studies have been done in the past on replication and stability of small plasmids in this bacterium; however, there are no reports on spatial localization and segregation of these plasmids. In the present study, a low copy number plasmid pDS3 containing pRC4 replicon was visualized in growing cells of Rhodococcus erythropolis PR4 (NBRC100887) using P1 parS-ParB-GFP system. Cells were initially cocci and then became rod shaped in exponential phase. Cocci cells were found to be non-replicating as evident by the presence of single fluorescence focus corresponding to the plasmid and diffuse fluorescence of DnaB-GFP. Rod shaped cells contained plasmid either present as one fluorescent focus observed at the cell center or two foci localized at quarter positions. The results suggest that the plasmid is replicated at the cell center and then it goes to quarter position. In order to observe the localization of plasmid with respect to nucleoid, plasmid segregation was also studied in filaments where it was found to be replicated at the cell center in a nucleoid free region. To the best of our knowledge, this is the first report on segregation of small plasmids in R. erythropolis. PMID:27935968

  10. Lipid storage in high-altitude Andean Lakes extremophiles and its mobilization under stress conditions in Rhodococcus sp. A5, a UV-resistant actinobacterium.

    PubMed

    Bequer Urbano, Susana; Albarracín, Virginia H; Ordoñez, Omar F; Farías, María E; Alvarez, Héctor M

    2013-03-01

    The production of triacylglycerols (TAG) or wax esters (WS) seems to be a widespread feature among extremophile bacteria living in high-altitude Andean Lakes (HAAL), Argentina. Twelve out of twenty bacterial strains isolated from HAAL were able to produce TAG or WS (between 2 and 17 % of cellular dry weight) under nitrogen-limiting culture conditions. Among these strains, the extremophile Rhodococcus sp. A5 accumulated significant amounts of TAG during growth on glucose (17 %, CDW) and hexadecane (32 %, CDW) as sole carbon sources. The role of accumulated TAG in the response to carbon starvation, osmotic stress, UV-radiation and desiccation was investigated in Rhodococcus sp. A5 using an inhibitor of TAG degradation. Cells degraded TAG during these stresses in the absence of the inhibitor. The inhibition of TAG mobilization affected cell survival during osmotic stress only during the initial growth stage. Little or no surviving cells were observed after carbon starvation, UV-treatment and desiccation, when TAG mobilization was inhibited. These results suggested that TAG metabolism is relevant for the adaptation and survival of A5 cells under carbon starvation, osmotic stress and UV irradiation, and essential under desiccation conditions, which prevail in HAAL environments.

  11. [Destruction of aromatic hydrocarbons by the Rhodococcus wratislaviensis KT112-7 strain isolated from waste products of a salt-mining factory].

    PubMed

    Egorova, D O; Korsakova, E S; Demakov, V A; Plotnikova, E G

    2013-01-01

    The destruction of aromatic hydrocarbons by the Rhodococcus wratislaviensis KT112-7 strain isolated from technogenic mineral waste products of the BKRU1 Uralkalii factory has been investigated (city of Berezniki, Perm krai). The R. wratislaviensis KT112-7 was shown to utilize increased concentrations of ophthalic (o-PA) (8 g/L) and benzoic (BA) (3.4 g/L) acids. The strain grows with o-FA, BA, and biphenyl at a NaCl content of up to 50, 90, and 75 g/L in the culture medium, respectively. Based on an analysis of the metabolic profile and nucleotide sequences of the bphA1, benA, and phtB genes, the KT112-7 strain was established to decompose o-PA via the formation of 3,4-dihydroxyphthalic and 3,4-dihydroxybenzoic acids. The decomposition of biphenyl is carried out via the formation of BA and then at low concentrations of NaCl (up to 50 g/L) via the formation of 4-hydroxybenzoic acid followed by its oxidation; at high concentrations of NaCl (over 60 g/L), via the direct oxidation of benzoic acid with the production of catechol. These data indicate that the Rhodococcus wratislaviensis KT112-7 destructor strain is a promising strain for the development of new biotechnologies directed at the utilization (transformation) of aromatic compounds, including under the conditions of increased mineralization.

  12. Endosulfan induced alteration in bacterial protein profile and RNA yield of Klebsiella sp. M3, Achromobacter sp. M6, and Rhodococcus sp. M2.

    PubMed

    Singh, Madhu; Singh, Dileep Kumar

    2014-01-30

    Three bacterial strains identified as Klebsiella sp. M3, Achromobacter sp. M6 and Rhodococcus sp. M2 were isolated by soil enrichment with endosulfan followed by shake flask enrichment technique. They were efficiently degrading endosulfan in the NSM (non sulfur medium) broth. Degradation of endosulfan was faster with the cell free extract of bacterial cells grown in the sulfur deficient medium (NSM) supplemented with endosulfan than that of nutrient rich medium (Luria Bertani). In the cell free extract of NSM supplemented with endosulfan as sole sulfur source, a unique band was visualized on SDS-PAGE but not with magnesium sulfate as the sole sulfur source in NSM and LB with endosulfan. Expression of a unique polypeptide band was speculated to be induced by endosulfan under sulfur starved condition. These unique polypeptide bands were identified as OmpK35 protein, sulfate binding protein and outer membrane porin protein, respectively, in Klebsiella sp. M3, Achromobacter sp. M6 and Rhodococcus sp. M2. Endosulfan showed dose dependent negative effect on total RNA yield of bacterial strains in nutrient rich medium. Absence of plasmid DNA indicated the presence of endosulfan metabolizing gene on genomic DNA. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Evaluation of equine breeding farm management and preventative health practices as risk factors for development of Rhodococcus equi pneumonia in foals.

    PubMed

    Chaffin, M Keith; Cohen, Noah D; Martens, Ronald J

    2003-02-15

    To determine whether foal management practices, environmental management, and preventative health practices are risk factors for development of Rhodococcus equi pneumonia in foals. Prospective matched case-control study. 2,764 foals on 64 equine breeding farms with 9,991 horses. During 1997, participating veterinarians completed paired data collection forms for comparison; 1 for an affected farm (containing > or = 1 foal with pneumonia caused by R equi) and 1 for a control farm. Information collected pertained to stabling facilities, environmental management, foal husbandry, and preventative equine health practices. Matched farm data compared by use of conditional logistic regression indicated that personnel on affected farms were more likely to attend foal births, test foals for adequacy of passive immunity, administer plasma or other treatments to foals to supplement serum immunoglobulin concentrations, administer hyperimmune plasma prophylactically to foals, vaccinate mares and foals against Streptococcus equi infection, and use multiple anthelmintics in deworming programs. Affected farms were also more likely to have foals that developed other respiratory tract disorders and were approximately 4 times as likely to have dirt floors in stalls used for housing foals as were control farms. Rhodococcus equi pneumonia does not appear to be associated with poor farm management or a lack of attention to preventative health practices. Housing foals in stalls with dirt floors may increase the risk for development of R equi pneumonia.

  14. Biotransformation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) by a Rabbit Liver Cytochrome P450: Insight into the Mechanism of RDX Biodegradation by Rhodococcus sp. Strain DN22

    PubMed Central

    Bhushan, Bharat; Trott, Sandra; Spain, Jim C.; Halasz, Annamaria; Paquet, Louise; Hawari, Jalal

    2003-01-01

    A unique metabolite with a molecular mass of 119 Da (C2H5N3O3) accumulated during biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 (D. Fournier, A. Halasz, J. C. Spain, P. Fiurasek, and J. Hawari, Appl. Environ. Microbiol. 68:166-172, 2002). The structure of the molecule and the reactions that led to its synthesis were not known. In the present study, we produced and purified the unknown metabolite by biotransformation of RDX with Rhodococcus sp. strain DN22 and identified the molecule as 4-nitro-2,4-diazabutanal using nuclear magnetic resonance and elemental analyses. Furthermore, we tested the hypothesis that a cytochrome P450 enzyme was responsible for RDX biotransformation by strain DN22. A cytochrome P450 2B4 from rabbit liver catalyzed a very similar biotransformation of RDX to 4-nitro-2,4-diazabutanal. Both the cytochrome P450 2B4 and intact cells of Rhodococcus sp. strain DN22 catalyzed the release of two nitrite ions from each reacted RDX molecule. A comparative study of cytochrome P450 2B4 and Rhodococcus sp. strain DN22 revealed substantial similarities in the product distribution and inhibition by cytochrome P450 inhibitors. The experimental evidence led us to propose that cytochrome P450 2B4 can catalyze two single electron transfers to RDX, thereby causing double denitration, which leads to spontaneous hydrolytic ring cleavage and decomposition to produce 4-nitro-2,4-diazabutanal. Our results provide strong evidence that a cytochrome P450 enzyme is the key enzyme responsible for RDX biotransformation by Rhodococcus sp. strain DN22. PMID:12620815

  15. The Steroid Catabolic Pathway of the Intracellular Pathogen Rhodococcus equi Is Important for Pathogenesis and a Target for Vaccine Development

    PubMed Central

    van der Geize, R.; Grommen, A. W. F.; Hessels, G. I.; Jacobs, A. A. C.; Dijkhuizen, L.

    2011-01-01

    Rhodococcus equi causes fatal pyogranulomatous pneumonia in foals and immunocompromised animals and humans. Despite its importance, there is currently no effective vaccine against the disease. The actinobacteria R. equi and the human pathogen Mycobacterium tuberculosis are related, and both cause pulmonary diseases. Recently, we have shown that essential steps in the cholesterol catabolic pathway are involved in the pathogenicity of M. tuberculosis. Bioinformatic analysis revealed the presence of a similar cholesterol catabolic gene cluster in R. equi. Orthologs of predicted M. tuberculosis virulence genes located within this cluster, i.e. ipdA (rv3551), ipdB (rv3552), fadA6 and fadE30, were identified in R. equi RE1 and inactivated. The ipdA and ipdB genes of R. equi RE1 appear to constitute the α-subunit and β-subunit, respectively, of a heterodimeric coenzyme A transferase. Mutant strains RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, were impaired in growth on the steroid catabolic pathway intermediates 4-androstene-3,17-dione (AD) and 3aα-H-4α(3′-propionic acid)-5α-hydroxy-7aβ-methylhexahydro-1-indanone (5α-hydroxy-methylhexahydro-1-indanone propionate; 5OH-HIP). Interestingly, RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, also displayed an attenuated phenotype in a macrophage infection assay. Gene products important for growth on 5OH-HIP, as part of the steroid catabolic pathway, thus appear to act as factors involved in the pathogenicity of R. equi. Challenge experiments showed that RE1ΔipdAB could be safely administered intratracheally to 2 to 5 week-old foals and oral immunization of foals even elicited a substantial protective immunity against a virulent R. equi strain. Our data show that genes involved in steroid catabolism are promising targets for the development of a live-attenuated vaccine against R. equi infections. PMID:21901092

  16. The steroid catabolic pathway of the intracellular pathogen Rhodococcus equi is important for pathogenesis and a target for vaccine development.

    PubMed

    van der Geize, R; Grommen, A W F; Hessels, G I; Jacobs, A A C; Dijkhuizen, L

    2011-08-01

    Rhodococcus equi causes fatal pyogranulomatous pneumonia in foals and immunocompromised animals and humans. Despite its importance, there is currently no effective vaccine against the disease. The actinobacteria R. equi and the human pathogen Mycobacterium tuberculosis are related, and both cause pulmonary diseases. Recently, we have shown that essential steps in the cholesterol catabolic pathway are involved in the pathogenicity of M. tuberculosis. Bioinformatic analysis revealed the presence of a similar cholesterol catabolic gene cluster in R. equi. Orthologs of predicted M. tuberculosis virulence genes located within this cluster, i.e. ipdA (rv3551), ipdB (rv3552), fadA6 and fadE30, were identified in R. equi RE1 and inactivated. The ipdA and ipdB genes of R. equi RE1 appear to constitute the α-subunit and β-subunit, respectively, of a heterodimeric coenzyme A transferase. Mutant strains RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, were impaired in growth on the steroid catabolic pathway intermediates 4-androstene-3,17-dione (AD) and 3aα-H-4α(3'-propionic acid)-5α-hydroxy-7aβ-methylhexahydro-1-indanone (5α-hydroxy-methylhexahydro-1-indanone propionate; 5OH-HIP). Interestingly, RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, also displayed an attenuated phenotype in a macrophage infection assay. Gene products important for growth on 5OH-HIP, as part of the steroid catabolic pathway, thus appear to act as factors involved in the pathogenicity of R. equi. Challenge experiments showed that RE1ΔipdAB could be safely administered intratracheally to 2 to 5 week-old foals and oral immunization of foals even elicited a substantial protective immunity against a virulent R. equi strain. Our data show that genes involved in steroid catabolism are promising targets for the development of a live-attenuated vaccine against R. equi infections.

  17. Optimizing Polychlorinated Biphenyl Degradation by Flavonoid-Induced Cells of the Rhizobacterium Rhodococcus erythropolis U23A.

    PubMed

    Pham, Thi Thanh My; Pino Rodriguez, Nancy Johanna; Hijri, Mohamed; Sylvestre, Michel

    2015-01-01

    There is evidence that many plant secondary metabolites may act as signal molecules to trigger the bacterial ability to metabolize polychlorinated biphenyls (PCBs) during the rhizoremediation process. However, the bases for the PCB rhizoremediation process are still largely unknown. The rhizobacterium Rhodococcus erythropolis U23A is unable to use flavanone as a growth substrate. However, on the basis of an assay that monitors the amount of 4-chlorobenzoate produced from 4-chlorobiphenyl by cells grown co-metabolically on flavanone plus sodium acetate, this flavonoid was previously found to be a potential inducer of the U23A biphenyl catabolic pathway. In this work, and using the same assay, we identified ten other flavonoids that did not support growth, but that acted as inducers of the U23A biphenyl pathway, and we confirmed flavonoid induction of the biphenyl catabolic pathway using quantitative real-time polymerase chain reaction (RT-qPCR) on the bphA gene. We also examined the effect of the growth co-substrate on flavonoid induction. Sodium acetate was replaced by glucose, mannose, sucrose, or mannitol, which are sugars found in plant root exudates. The data showed that the level of induction of strain U23A biphenyl-degrading enzymes was significantly influenced by the nature and concentration of the flavonoid in the growth medium, as well as by the substrate used for growth. Sucrose allowed for an optimal induction response for most flavonoids. Some flavonoids, such as flavone and isoflavone, were better inducers of the biphenyl catabolic enzymes than biphenyl itself. We also found that all flavonoids tested in this work were metabolized by strain U23A during co-metabolic growth, but that the metabolite profiles, as well as the level of efficiency of degradation, differed for each flavonoid. To obtain insight into how flavonoids interact with strain U23A to promote polychlorinated biphenyl (PCB) degradation, we determined the concentration of flavanone at

  18. Molecular characterization of a lipid-modified virulence-associated protein of Rhodococcus equi and its potential in protective immunity.

    PubMed Central

    Tan, C; Prescott, J F; Patterson, M C; Nicholson, V M

    1995-01-01

    Virulent strains of Rhodococcus equi produce plasmid-mediated 15- and 17-kDa proteins, which are thermoregulated and apparently surface-expressed. We demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that R. equi produce three antigenically-related virulence-associated proteins, a diffuse 18-22-kDa, a 17.5-kDa and a 15-kDa protein. Phase partitioning of whole cells of R. equi strain 103 with Triton X-114 (TX-114) and labelling with [3H]-labelled palmitic acid showed that the two higher molecular weight proteins are hydrophobic and lipid modified. The 15-kDa protein did not partition into TX-114 and was not lipid modified. Cloning and expression of a fragment of the R. equi virulence plasmid in Escherichia coli showed that the three proteins were expressed from a single gene. Sequence analysis of this gene (designated vapA) revealed a 570-bp open reading frame encoding a polypeptide of 189 amino acids with a calculated molecular mass of 19,175 Da. The mature, nonlipid modified protein had a calculated mass of 16,246 Da. The 17.5- and 18-22-kDa forms of the protein are therefore due to lipid modification. No significant sequence homology of the vapA gene with other reported nucleotide sequences were found. Opsonization of virulent R. equi with an IgG1 mouse monoclonal antibody (MAb103) to the VapA protein significantly enhanced uptake in the murine macrophage cell line IC-21. Intraperitoneal injection of mice with Mab103 enhanced initial clearance from the liver of mice challenged intravenously with R. equi. Immunization of mice with the lipid-modified VapA purified by SDS-PAGE fractionation or with acetone precipitated VapA protein following TX-114 extraction resulted in significantly enhanced clearance from the liver and spleen following intravenous challenge. The VapA protein of R. equi appears therefore to be a protective immunogen. Images Fig. 1. Fig. 4. PMID:7704843

  19. Genome and Proteome Analysis of Rhodococcus erythropolis MI2: Elucidation of the 4,4´-Dithiodibutyric Acid Catabolism

    PubMed Central

    Khairy, Heba; Meinert, Christina; Wübbeler, Jan Hendrik; Poehlein, Anja; Daniel, Rolf; Voigt, Birgit; Riedel, Katharina; Steinbüchel, Alexander

    2016-01-01

    Rhodococcus erythropolis MI2 has the extraordinary ability to utilize the xenobiotic 4,4´-dithiodibutyric acid (DTDB). Cleavage of DTDB by the disulfide-reductase Nox, which is the only verified enzyme involved in DTDB-degradation, raised 4-mercaptobutyric acid (4MB). 4MB could act as building block of a novel polythioester with unknown properties. To completely unravel the catabolism of DTDB, the genome of R. erythropolis MI2 was sequenced, and subsequently the proteome was analyzed. The draft genome sequence consists of approximately 7.2 Mbp with an overall G+C content of 62.25% and 6,859 predicted protein-encoding genes. The genome of strain MI2 is composed of three replicons: one chromosome and two megaplasmids with sizes of 6.45, 0.4 and 0.35 Mbp, respectively. When cells of strain MI2 were cultivated with DTDB as sole carbon source and compared to cells grown with succinate, several interesting proteins with significantly higher expression levels were identified using 2D-PAGE and MALDI-TOF mass spectrometry. A putative luciferase-like monooxygenase-class F420-dependent oxidoreductase (RERY_05640), which is encoded by one of the 126 monooxygenase-encoding genes of the MI2-genome, showed a 3-fold increased expression level. This monooxygenase could oxidize the intermediate 4MB into 4-oxo-4-sulfanylbutyric acid. Next, a desulfurization step, which forms succinic acid and volatile hydrogen sulfide, is proposed. One gene coding for a putative desulfhydrase (RERY_06500) was identified in the genome of strain MI2. However, the gene product was not recognized in the proteome analyses. But, a significant expression level with a ratio of up to 7.3 was determined for a putative sulfide:quinone oxidoreductase (RERY_02710), which could also be involved in the abstraction of the sulfur group. As response to the toxicity of the intermediates, several stress response proteins were strongly expressed, including a superoxide dismutase (RERY_05600) and an osmotically induced

  20. Optimizing Polychlorinated Biphenyl Degradation by Flavonoid-Induced Cells of the Rhizobacterium Rhodococcus erythropolis U23A

    PubMed Central

    Hijri, Mohamed; Sylvestre, Michel

    2015-01-01

    There is evidence that many plant secondary metabolites may act as signal molecules to trigger the bacterial ability to metabolize polychlorinated biphenyls (PCBs) during the rhizoremediation process. However, the bases for the PCB rhizoremediation process are still largely unknown. The rhizobacterium Rhodococcus erythropolis U23A is unable to use flavanone as a growth substrate. However, on the basis of an assay that monitors the amount of 4-chlorobenzoate produced from 4-chlorobiphenyl by cells grown co-metabolically on flavanone plus sodium acetate, this flavonoid was previously found to be a potential inducer of the U23A biphenyl catabolic pathway. In this work, and using the same assay, we identified ten other flavonoids that did not support growth, but that acted as inducers of the U23A biphenyl pathway, and we confirmed flavonoid induction of the biphenyl catabolic pathway using quantitative real-time polymerase chain reaction (RT-qPCR) on the bphA gene. We also examined the effect of the growth co-substrate on flavonoid induction. Sodium acetate was replaced by glucose, mannose, sucrose, or mannitol, which are sugars found in plant root exudates. The data showed that the level of induction of strain U23A biphenyl-degrading enzymes was significantly influenced by the nature and concentration of the flavonoid in the growth medium, as well as by the substrate used for growth. Sucrose allowed for an optimal induction response for most flavonoids. Some flavonoids, such as flavone and isoflavone, were better inducers of the biphenyl catabolic enzymes than biphenyl itself. We also found that all flavonoids tested in this work were metabolized by strain U23A during co-metabolic growth, but that the metabolite profiles, as well as the level of efficiency of degradation, differed for each flavonoid. To obtain insight into how flavonoids interact with strain U23A to promote polychlorinated biphenyl (PCB) degradation, we determined the concentration of flavanone at

  1. Formation and resuscitation of "non-culturable" cells of Rhodococcus rhodochrous and Mycobacterium tuberculosis in prolonged stationary phase.

    PubMed

    Shleeva, M O; Bagramyan, K; Telkov, M V; Mukamolova, G V; Young, M; Kell, D B; Kaprelyants, A S

    2002-05-01

    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

  2. Short report: Identification of virulence-associated plasmids in Rhodococcus equi in humans with and without acquired immunodeficiency syndrome in Brazil.

    PubMed

    Ribeiro, Márcio Garcia; Takai, Shinji; de Vargas, Agueda Castagna; Mattos-Guaraldi, Ana Luiza; Ferreira Camello, Thereza Cristina; Ohno, Ryoko; Okano, Hajime; Silva, Aristeu Vieira da

    2011-09-01

    Virulence of Rhodococcus equi strains from 20 humans in Brazil was investigated by using a polymerase chain reaction to characterize isolates as virulent (VapA), intermediately virulent (VapB), and avirulent. Nine isolates were obtained from human immunodeficiency virus (HIV)-positive patients, six from HIV-negative patients, and five from patients of unknown status. Five isolates were VapB positive, four were VapA positive, and eleven were avirulent. Among the nine isolates from HIV-positive patients, five contained VapB plasmids and two contained VapA plasmids. Five VapB-positive isolates had the type 8 virulence plasmid. Eleven of the patients had a history of contact with livestock and/or a farm environment, and none had contact with pigs.

  3. Transcriptional regulation by VirR and VirS of members of the Rhodococcus equi virulence-associated protein multigene family.

    PubMed

    Kakuda, Tsutomu; Miyazaki, Shiko; Hagiuda, Hirofumi; Takai, Shinji

    2015-08-01

    A virulence plasmid of Rhodococcus equi harbors the vap mutigene family. Here it is shown that transcription of vap gene family members other than vapA (vapD, vapE and vapG) is regulated by temperature and pH and abolished when either virS or virR is deleted. Expression of VirS in the absence of functional VirR was found to increase the transcription of vap genes to the amount expressed in the presence of VirR. These findings suggest that transcription of vap genes is regulated by VirS and that VirR is involved in the mechanism of transcriptional responses to temperature and pH. © 2015 The Societies and Wiley Publishing Asia Pty Ltd.

  4. Isolation of a novel carotenoid, OH-chlorobactene glucoside hexadecanoate, and related rare carotenoids from Rhodococcus sp. CIP and their antioxidative activities.

    PubMed

    Osawa, Ayako; Kasahara, Asami; Mastuoka, Shoko; Gassel, Sören; Sandmann, Gerhard; Shindo, Kazutoshi

    2011-01-01

    In the course of screening for antioxidative carotenoids from bacteria, we isolated and identified a novel carotenoid, OH-chlorobactene glucoside hexadecanoate (4), and rare carotenoids, OH-chlorobactene glucoside (1), OH-γ-carotene glucoside (2) and OH-4-keto-γ-carotene glucoside hexadecanoate (3) from Rhodococcus sp. CIP. The singlet oxygen ((1)O(2)) quenching model of these carotenoids showed potent antioxidative activities IC(50) 14.6 µM for OH-chlorobactene glucoside hexadecanoate (4), 6.5 µM for OH-chlorobactene glucoside (1), 9.9 µM for OH-γ-carotene glucoside (2) and 7.3 µM for OH-4-keto-γ-carotene glucoside hexadecanoate (3).

  5. Effects of a static magnetic field on phenol degradation effectiveness and Rhodococcus erythropolis growth and respiration in a fed-batch reactor.

    PubMed

    Křiklavová, Lucie; Truhlář, Martin; Škodováa, Petra; Lederer, Tomáš; Jirků, Vladimír

    2014-09-01

    The aim of this study was to evaluate the impact of short-term repeated exposure to a static magnetic field (induction 370 mT) on the Rhodococcus erythropolis cells. Specifically, it was ascertained the magnetic field's potential to influence degradation of a phenol substrate, cell growth and respiration activity (oxygen consumption) during substrate biodegradation. The experiment took place over 3 days, with R. erythropolis exposed to the magnetic field for the first day. During the experiment, different recirculation rates between the reactor and the magnetic contactor has been tested. Use of the magnetic field at higher recirculation rates (residence time in contactor was less than 7 min) stimulated substrate (phenol) oxidation by around 34%; which, in turn, promoted R. erythropolis growth by around 28% by shortening the lag- and exponential-phases and increasing bacterial respiration activity by around 10%.

  6. An amino acid at position 142 in nitrilase from Rhodococcus rhodochrous ATCC 33278 determines the substrate specificity for aliphatic and aromatic nitriles

    PubMed Central

    Yeom, Soo-Jin; Kim, Hye-Jung; Lee, Jung-Kul; Kim, Dong-Eun; Oh, Deok-Kun

    2008-01-01

    Nitrilase from Rhodococcus rhodochrous ATCC 33278 hydrolyses both aliphatic and aromatic nitriles. Replacing Tyr-142 in the wild-type enzyme with the aromatic amino acid phenylalanine did not alter specificity for either substrate. However, the mutants containing non-polar aliphatic amino acids (alanine, valine and leucine) at position 142 were specific only for aromatic substrates such as benzonitrile, m-tolunitrile and 2-cyanopyridine, and not for aliphatic substrates. These results suggest that the hydrolysis of substrates probably involves the conjugated π-electron system of the aromatic ring of substrate or Tyr-142 as an electron acceptor. Moreover, the mutants containing charged amino acids such as aspartate, glutamate, arginine and asparagine at position 142 displayed no activity towards any nitrile, possibly owing to the disruption of hydrophobic interactions with substrates. Thus aromaticity of substrate or amino acid at position 142 in R. rhodochrous nitrilase is required for enzyme activity. PMID:18412544

  7. Cloning of an ORF with homology to Mycobacterium echA1, encoding the enoyl-CoA hydratase, in Rhodococcus fascians.

    PubMed

    Humanes, L; García-Fernández, J M; Roldán, J M; Diez, J

    1999-01-01

    An open reading frame encoding a polypeptide of significant homology (55.7% identity) with the enoyl-CoA hydratase encoded by the gene echA1 from Mycobacterium tuberculosis has been found in the genome of the plant-pathogen bacteria Rhodococcus fascians strain NRRL-B-15096. Sequence alignments showed that it possesses several conserved blocks common to E. coli, M. tuberculosis and human mitochondria. One of such blocks includes a glutamate residue located at position 149, corresponding to the glutamate 139 of Escherichia coli. This glutamate was previously shown to be the catalytic residue of enoyl-CoA hydratase in the multienzyme complex of fatty acid oxidation from E. coli. Our results provide additional information on the conserved domains of this enzyme. Significant homologies in other genome regions between R. fascians and M. tuberculosis confirm their phylogenetic relationship.

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

    PubMed

    Nagy, I; Schoofs, G; De Schrijver, A; Vanderleyden, J; De Mot, R

    1997-07-01

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

  9. Biodegradation of methylthio-s-triazines by Rhodococcus sp. strain FJ1117YT, and production of the corresponding methylsulfinyl, methylsulfonyl and hydroxy analogues.

    PubMed

    Fujii, Kunihiko; Takagi, Kazuhiro; Hiradate, Syuntaro; Iwasaki, Akio; Harada, Naoki

    2007-03-01

    A novel bacterial strain FJ1117YT was isolated from an enrichment culture with the herbicide simetryn. The isolate was capable of degrading the herbicide supplied as the sole sulfur source in an aquatic batch culture. The strain FJ1117YT was identified as that belonging to Rhodococcus sp. on the basis of comparative morphology, physiological characteristics and comparison of the 16S rRNA gene sequence. The biodegradation pathway of simetryn was established by isolating the methylsulfinyl analogue as the first metabolite and by identification of the methylsulfonyl intermediate and the hydroxy analogue by liquid chromatography-mass spectrometry (LC-MS) and/or nuclear magnetic resonance (NMR) analysis. The results indicate that the methylthio group was progressively oxidised and hydrolysed by the strain FJ1117YT. The same strain is also able to metabolise other methylthio-s-triazines such as ametryn, desmetryn, dimethametryn and prometryn through similar pathways.

  10. Movement disorders in encephalitis induced by Rhodococcus aurantiacus infection relieved by the administration of L-dopa and anti-T-cell antibodies

    PubMed Central

    Min, Y; Asano, M; Kohanawa, M; Minagawa, T

    1999-01-01

    Mice injected with Rhodococcus aurantiacus by the intravenous (i.v.) route show neurological disorders, hemiparesis, vertical headshake and turn-round gait after day 7 postinfection (p.i.). Neurological symptoms caused by i.v. inoculation of R. aurantiacus were relieved by treatment with levodopa (l-dopa). R. aurantiacus was isolated from the brain and was found to be completely eliminated at day 7 p.i. Focal encephalitis was mainly observed in the brain stem, and T cells could be isolated from the brain after day 7 p.i. Administration of both an anti-CD4 monoclonal antibody (mAb) and an anti-CD8 mAb suppressed neurological symptoms. These results suggest that R. aurantiacus induces movement disorders in mice, and that the symptoms are mediated by T cells infiltrating the brain, rather than directly by the bacterium. PMID:10233672

  11. [Synthesis of surfactants by Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV B-7241 and Nocardia vaccinii IMV B-7405 on industrial waste].

    PubMed

    Pirog, T P; Sofilkanich, A P; Pokora, K A; Shevchuk, T A; Iutinskaia, G A

    2014-01-01

    The synthesis of surfactants by Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV B-7241 and Nocardia vaccinii IMV B-7405 on industrial waste (food and oil-processing industry, production of biodiesel) was investigated. The possibility of replacing the expensive substrates (n-hexadecane and ethanol) by industrial waste (oil and fat industry, fried sunflower oil, glycerol, liquid paraffin) for the surfactant biosynthesis was established. The conditional concentration of surfactants was maximal on oil containing substrates and exceeded those on n-hexadecane and ethanol 2-3 times. The highest rates of surfactants synthesis were observed on fried sunflower oil with the use of inoculum grown on carbohydrate substrates (glucose, molasses). It was established that the addition of glucose (0.1%) was accompanied by 2-4-fold intensification of surfactants synthesis by R. erythropolis IMV Ac-5017 and N. vaccinii IMV B-7405 on fried sunflower oil (2%).

  12. Heterologous production of kasugamycin, an aminoglycoside antibiotic from Streptomyces kasugaensis, in Streptomyces lividans and Rhodococcus erythropolis L-88 by constitutive expression of the biosynthetic gene cluster.

    PubMed

    Kasuga, Kano; Sasaki, Akira; Matsuo, Takashi; Yamamoto, Chika; Minato, Yuiko; Kuwahara, Naoya; Fujii, Chikako; Kobayashi, Masayuki; Agematu, Hitosi; Tamura, Tomohiro; Komatsu, Mamoru; Ishikawa, Jun; Ikeda, Haruo; Kojima, Ikuo

    2017-02-27

    Kasugamycin (KSM), an aminoglycoside antibiotic isolated from Streptomyces kasugaensis cultures, has been used against rice blast disease for more than 50 years. We cloned the KSM biosynthetic gene (KBG) cluster from S. kasugaensis MB273-C4 and constructed three KBG cassettes (i.e., cassettes I-III) to enable heterologous production of KSM in many actinomycetes by constitutive expression of KBGs. Cassette I comprised all putative transcriptional units in the cluster, but it was placed under the control of the P neo promoter from Tn5. It was not maintained stably in Streptomyces lividans and did not transform Rhodococcus erythropolis. Cassette II retained the original arrangement of KBGs, except that the promoter of kasT, the specific activator gene for KBG, was replaced with P rpsJ , the constitutive promoter of rpsJ from Streptomyces avermitilis. To enhance the intracellular concentration of myo-inositol, an expression cassette of ino1 encoding the inositol-1-phosphate synthase from S. avermitilis was inserted into cassette II to generate cassette III. These two cassettes showed stable maintenance in S. lividans and R. erythropolis to produce KSM. Particularly, the transformants of S. lividans induced KSM production up to the same levels as those produced by S. kasugaensis. Furthermore, cassette III induced more KSM accumulation than cassette II in R. erythropolis, suggesting an exogenous supply of myo-inositol by the ino1 expression in the host. Cassettes II and III appear to be useful for heterologous KSM production in actinomycetes. Rhodococcus exhibiting a spherical form in liquid cultivation is also a promising heterologous host for antibiotic fermentation.

  13. Molecular characterization of Rhodococcus equi isolates from horses in Poland: pVapA characteristics and plasmid new variant, 85-kb type V.

    PubMed

    Witkowski, Lucjan; Rzewuska, Magdalena; Takai, Shinji; Chrobak-Chmiel, Dorota; Kizerwetter-Świda, Magdalena; Feret, Małgorzata; Gawryś, Marta; Witkowski, Maciej; Kita, Jerzy

    2017-01-26

    Rhodococcus equi is one of the most significant bacterial pathogens affecting foals up to 6 months of age worldwide. Rhodococcosis is present in Poland however information about molecular characterization of R. equi isolates is scarce. This study describes molecular characterization of Rhodococcus equi infection on 13 horse breeding farms in Poland between 2001 and 2012. Samples were collected by tracheobronchial aspiration from pneumonic foals or during necropsy. The R. equi isolates were genotyped by plasmid profiling and pulsed-field gel electrophoresis. Totally, 58 R. equi isolates were investigated. One isolate lost its plasmid. Among the 57 VapA-positive isolates, 48 contained 85-kb type I plasmid (82.8%), 8 contained 87-kb type I plasmid (13.8%). One isolate (1.7%) had a unique restriction cleavage pattern and the 2nd fragment of EcoRI digests of this plasmid DNA was about 2600 bases smaller than that of the 85 kb type I. This new plasmid variant was designated as the "85-kb type V". Among the 58 isolates typeable with VspI-PFGE, ten PFGE clusters were detected. The majority of foals were infected mostly with isolates of low genetic diversity. Most of clinical isolates of R. equi from foals in Poland contain pVapA 85-kb type I and 87-kb type I similarly to the other European countries and the United States. However, the new variant of pVapA 85-kb type V was identified. The chromosomal variability was detected among some of the investigated isolates and the presence of farm-specific isolates might be possible.

  14. On the Kinetic and Allosteric Regulatory Properties of the ADP-Glucose Pyrophosphorylase from Rhodococcus jostii: An Approach to Evaluate Glycogen Metabolism in Oleaginous Bacteria

    PubMed Central

    Cereijo, Antonela E.; Asencion Diez, Matías D.; Dávila Costa, José S.; Alvarez, Héctor M.; Iglesias, Alberto A.

    2016-01-01

    Rhodococcus spp. are oleaginous bacteria that accumulate glycogen during exponential growth. Despite the importance of these microorganisms in biotechnology, little is known about the regulation of carbon and energy storage, mainly the relationship between glycogen and triacylglycerols metabolisms. Herein, we report the molecular cloning and heterologous expression of the gene coding for ADP-glucose pyrophosphorylase (EC 2.7.7.27) of Rhodococcus jostii, strain RHA1. The recombinant enzyme was purified to electrophoretic homogeneity to accurately characterize its oligomeric, kinetic, and regulatory properties. The R. jostii ADP-glucose pyrophosphorylase is a homotetramer of 190 kDa exhibiting low basal activity to catalyze synthesis of ADP-glucose, which is markedly influenced by different allosteric effectors. Glucose-6P, mannose-6P, fructose-6P, ribose-5P, and phosphoenolpyruvate were major activators; whereas, NADPH and 6P-gluconate behaved as main inhibitors of the enzyme. The combination of glucose-6P and other effectors (activators or inhibitors) showed a cross-talk effect suggesting that the different metabolites could orchestrate a fine regulation of ADP-glucose pyrophosphorylase in R. jostii. The enzyme exhibited some degree of affinity toward ATP, GTP, CTP, and other sugar-1P substrates. Remarkably, the use of glucosamine-1P was sensitive to allosteric activation. The relevance of the fine regulation of R. jostii ADP-glucose pyrophosphorylase is further analyzed in the framework of proteomic studies already determined for the bacterium. Results support a critical role for glycogen as a temporal reserve that provides a pool of carbon able of be re-routed to produce long-term storage of lipids under certain conditions. PMID:27313571

  15. Rhodococcus sp. RB1 grows in the presence of high nitrate and nitrite concentrations and assimilates nitrate in moderately saline environments.

    PubMed

    Blasco, R; Martínez-Luque, M; Madrid, M P; Castillo, F; Moreno-Vivián, C

    2001-06-01

    Rhodococcus sp. RB1 was able to thrive in media with up to 0.9 M NaCl or KCl and in the presence of high concentrations of nitrate (up to 0.9 M) and nitrite (up to 60 mM), but only under oxic conditions. An adaptation period was not required for salt tolerance, but a rapid extrusion of K+ and intake of Na+ was observed after addition of 0.5 M NaCl. Nitrate assimilation was limited by the carbon supply, but nitrite was not accumulated in the culture medium, even at nitrate concentrations as high as 0.8 M, thus suggesting that nitrite reduction does not limit nitrate assimilation. The presence of NaCl or KCl did not affect nitrate or nitrite uptake, which were completely inhibited by ammonium or glutamine. Rhodococcus sp. RB1 nitrate reductase had an apparent molecular mass of 142 kDa and used NADH and reduced bromophenol blue or viologens as electron donors, independently of the presence of salt. The enzyme was associated with an NADH-diaphorase activity and was induced by nitrate and repressed by ammonium or glutamine, thus showing typical biochemical and regulatory properties of bacterial assimilatory NADH-nitrate reductases. The enzyme was active in vitro in the presence of 3 M NaCl or KCI, but the maximal activity was observed at 0.5 M salt. Addition of 2 M NaCl increased the optimal temperature of the enzyme from 12 to 32 degrees C, but the optimal pH (10.3) was unaffected.

  16. Purification and characterization of catechol 1,2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 and cloning and sequencing of its catA gene.

    PubMed Central

    Strachan, P D; Freer, A A; Fewson, C A

    1998-01-01

    A method was developed for the purification of catechol 1, 2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 that had been grown in the presence of benzyl alcohol. The enzyme has very similar apparent Km (1-2 microM) and Vmax (13-19 units/mg of protein) values for the intradiol cleavage of catechol, 3-methylcatechol and 4-methylcatechol and it is optimally active at pH9. Cross-linking studies indicate that the enzyme is a homodimer. It contains 0.6 atoms of Fe per subunit. The enzyme was crystallized with 15% (w/v) poly(ethylene glycol) 4000/0.33 M CaCl2/25 mM Tris (pH7.5) by using a microseeding technique. Preliminary X-ray characterization showed that the crystals are in space group C2 with unit-cell dimensions a=111.9 A, b=78.1 A, c=134.6 A, beta=100 degrees. An oligonucleotide probe, made by hemi-nested PCR, was used to clone the gene encoding catechol 1,2-dioxygenase (catA). The deduced 282-residue sequence corresponds to a protein of molecular mass 31539 Da, close to the molecular mass of 31558 Da obtained by electrospray MS of the purified enzyme. catA was subcloned into the expression vector pTB361, allowing the production of catechol 1,2-dioxygenase to approx. 40% of the total cellular protein. The deduced amino acid sequence of the enzyme has 56% and 75% identity with the catechol 1, 2-dioxygenases of Arthrobacter mA3 and Rhodococcus erythropolis AN-13 respectively, but less than 35% identity with intradiol catechol and chlorocatechol dioxygenases of Gram-negative bacteria. PMID:9677336

  17. A 3-(3-hydroxyphenyl)propionic acid catabolic pathway in Rhodococcus globerulus PWD1: cloning and characterization of the hpp operon.

    PubMed Central

    Barnes, M R; Duetz, W A; Williams, P A

    1997-01-01

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