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DI, CANITO ALESSANDRA. « Genomic and functional analysis of Rhodococcus strains to identify genes and degradative functions for soil quality evaluation ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2019. http://hdl.handle.net/10281/241307.
Texte intégralRésumé :
La qualità del suolo è una delle principali problematiche ambientali degli ultimi decenni a causa dell’aumento dell’inquinamento antropico. In condizioni di stress, i microrganismi del suolo subiscono alterazioni che attraverso tecnologie molecolari possono essere usate come parametro per il monitoraggio dei siti contaminati. I batteri appartenenti al genere Rhodococcus hanno un ruolo importante nella degradazione dei composti più recalcitranti. Sono versatili ed ampiamente distribuiti in natura; essi degradano diversi composti organici, tra cui idrocarburi alifatici ed aromatici, eterociclici, nitrili, sulfuri ed erbicidi. Inoltre, essi possono sopravvivere in presenza di composti tossici, carenza di carbonio, irradiazione UV e stress osmotico. Questa versatilità è correlata alla complessità dei loro genomi, i quali contengono molteplici geni catabolici, ridondanza genica e un sofisticato network regolatorio. L’obiettivo di questo progetto è ottenere nuovi tools molecolari da ceppi di Rhodococcus da usare come marcatori per valutare la qualità dei suoli, mediante analisi dei pathway metabolici e dei cluster genici coinvolti nella degradazione dei contaminanti ambientali. In questo lavoro, l’attenzione è stata rivolta verso i genomi dei ceppi: R. opacus R7, R. aetherivorans BCP1 e R. erythropolis MI2. Un’analisi fenotipica ha permesso di valutare il potenziale metabolico e la risposta allo stress dei ceppi R7 e BCP1; sono stati testati diversi contaminanti (idrocarburi alifatici e cicloalcani, aromatici, policiclici aromatici, acidi naftenici ed altri acidi carbossilici) e varie condizioni di stress (alta osmolarità, differenti valori di pH, composti tossici, antibiotici). Un approccio genomico ha permesso di correlare le abilità metaboliche a determinanti genici, coinvolti nei diversi metabolismi (naftalene, o-xilene, n-alcani, acidi naftenici, fenoli, ftalato) e nella persistenza ambientale. In particolare, sono stati esaminati i pathway degradativi dell’o-xilene e degli acidi naftenici di R. opacus R7. Analisi bioinformatiche e molecolari hanno permesso di valutare il coinvolgimento di diversi geni nei pathway degradativi. R7 è in grado di degradare l’o-xilene inducendo la trascrizione dei geni akb (sistema diossigenasico) formando il diidrodiolo. Tuttavia, la ridondanza di monossigenasi e idrossilasi (prmA and pheA1A2A3), ha suggerito l’attivazione di altri sistemi convergenti, strategia utilizzata dai rhodococci per degradare composti recalcitranti e persistere in ambienti contaminati. I pathway degradativi degli acidi naftenici (NAs) non sono ancora noti ma sono state proposte due possibili vie: i) aromatizzazione dell’anello del cicloesano ii) attivazione come CoA tioestere. I risultati delle RT e RT-qPCR hanno mostrato che R7 degrada l’acido cicloesanocarbossilico (CHCA), attraverso una cicloesano carbossilato-CoA ligasi (aliA). L’applicazione di questo lavoro è stata dimostrata in esperimenti di microcosmo simulando condizioni reali con sabbia bioaugmentata con R7. Le capacità dei batteri autoctoni e di R7 di degradare il CHCA sono state comparate e i risultati mostrano che R7 degrada il contaminante più velocemente rispetto alla comunità microbica e che il suo contributo aumenta la velocità di degradazione del CHCA, seguita monitorando l’espressione del gene aliA mediante esperimenti di RT e RT-qPCR. Un’applicazione biotecnologica di questo lavoro è stata valutata in R. erythropolis MI2, studiando il pathway di degradazione del 4,4’- acido disolfuro ditiobutirrico (DTDB), un promettente substrato per la sintesi dei politioesteri poiché il suo intermedio metabolico, acido 4-mercaptobutirrico ne è un precursore. L’obiettivo di questo studio è stato perseguito generando mutanti di delezione del ceppo MI2 per i geni coinvolti nelle reazioni finali del pathway di degradazione.Soil quality has been one of the major issues of the last decades, because of the increase of anthropogenic pollution. Soil contains organisms involved in vital functions (nutrient/hydrological cycles and degradation of toxic compounds). Under stress conditions, soil microorganisms undergo several alterations so molecular technologies use microbial communities as an ecological parameter in monitoring polluted sites. Bacteria belonging to Rhodococcus genus have an important role in recalcitrant compound degradations. It is a metabolically versatile genus, widely distributed in nature. Rhodococcus spp. can degrade a wide range of organic compounds (aliphatic/aromatic hydrocarbons, heterocyclic, nitriles, sulfuric, herbicides) and to survive in presence of toxic compounds, carbon starvation, UV irradiation and osmotic stress. In line with their catabolic diversity, they possess large and complex genomes, containing a multiplicity of catabolic genes, high genetic redundancy and a sophisticated regulatory network. The aim of this project is to obtain molecular tools to use as "marker" sequences for soil assessment, through analysis of metabolic pathways and catabolic gene clusters involved in the degradation of the most diffused environmental contaminants. In particular, this work focused the attention on three Rhodococcus strain genomes: R. opacus R7, R. aetherivorans BCP1 and R. erythropolis MI2. A Phenotype Microarray approach was used to evaluate R7 and BCP1 strains metabolic potential and their stress response. Also, the capability to utilize various contaminants (aliphatic hydrocarbons and cycloalkanes, aromatic compounds, polycyclic aromatic compounds, naphthenic acids and other carboxylic acids) and to persist under stress conditions (high osmolarity, pH stress, toxic compounds, antibiotics) was tested. A genome-based approach was used to relate their abilities to genetic determinants involved in the analysed metabolisms (naphthalene, o-xylene, n-alkanes, naphthenic acids, phenols, phthalate) and in their environmental persistence. In particular, o-xylene and naphthenic acids degradations were investigated in R. opacus R7. Computational and molecular analyses revealed the putative involvement of several genes in these degradation pathways. R7 can degrade o-xylene by the induction of the akb genes (deoxygenation) producing the corresponding dihydrodiol. Likewise, the redundancy of sequences encoding for monooxygenases/hydroxylases (prmA and pheA1A2A3), supports the involvement of other genes that induce the formation of phenols, converging to the phenol oxidation path. The activation of converging oxygenase systems represents a strategy in Rhodococcus genus to degrade recalcitrant compounds and to persist in contaminated environments. NAs degradation pathway is not fully clear but two main routes have been proposed: i) aromatization of the cyclohexane ring ii) activation as CoA thioester. RT and RT-qPCR results showed that R. opacus R7 degrade cyclohexanecarboxylic acid (CHCA) molecule (used as a model) by a cyclohexane carboxylate CoA ligase (aliA). An application of this work was demonstrated by a microcosm approach, simulating a bioaugmentation process with R7 strain. Autochthone bacteria and R7 capabilities to degrade CHCA were evaluated and compared; results indicated that R7 can degrade the contaminant faster than the microbial community and that its contribute increased CHCA degradation rate. The degradation rate was followed by RT and RT-qPCR, monitoring the expression of the aliA gene. Moreover, a biotechnological application was investigated in R. erythropolis MI2, studying the disulfide 4,4-dithiodibutyric acid (DTDB) degradation pathway. DTDB is a promising substrate for polythioester (PTE) synthesis; indeed, its degradation produces the PTE building block 4-mercaptobutyric acid. The aim was pursued generating R. erythropolis MI2 marker-free deletion mutants for genes involved in the final steps of the pathway.
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Naʼamnieh, Shukrallah. « Entwicklung eines rekombinanten Ganzzellsystems - Klonierung, Coexpression und Mutagenese der Phenylalanin-Dehydrogenase aus Rhodococcus sp. M4 und des malic enzymes aus E.coli K12 ». [S.l. : s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=966050142.
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Abokitse, Kofi. « Biochemische und molekularbiologische Charakterisierung von Alkoholdehydrogenasen und einer Oxygenase aus Rhodococcus Spezies ». [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=971968446.
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Stoecker, Matthew A. « Biodegradation of aromatic and aliphatic hydrocarbons by Rhodococcus spp. / ». Thesis, Connect to this title online ; UW restricted, 1998. http://hdl.handle.net/1773/11495.
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Ganguly, Sangeeta. « Enhanced Stabilization of Nitrile Hydratase Enzyme From Rhodococcus Sp. DAP 96253 and Rhodococcus ». Digital Archive @ GSU, 2007. http://digitalarchive.gsu.edu/biology_diss/25.
Texte intégralRésumé :
Treatment of industrial wastewaters contaminated with toxic and hazardous organics can be a costly process. In the case of acrylonitrile production, due to highly volatile and toxic nature of the contaminant organics, production wastewaters are currently disposed by deepwell injection without treatment. Under the terms granting deepwell injection of the waste, alternative treatments must be investigated, and an effective treatment identified. Cells of two Gram-positive bacteria, Rhodococcus sp. DAP 96253 and R. rhodochrous DAP 96622 were evaluated for their potential as biocatalysts for detoxification of acrylonitrile production wastewaters. Rhodococcus sp. DAP 96253 and R. rhodochrous DAP 96622 when multiply induced, are capable of utilizing the hazardous nitrile and amide components present in the wastewater as sole carbon and/or nitrogen sources, employing a 2-step enzymatic system involving nitrile hydratase (NHase) and amidase enzymes. There is a significant potential for overproduction of NHase upon multiple induction. However, high-level multiple induction required the presence of highly toxic nitriles and/or amides in the growth medium. Asparagine and glutamine were identified as potent inducers with overexpression at 40% of total soluble cellular protein as NHase. In native form (either cell free enzymes or whole cells) the desired NHase is very labile. In order to develop a practical catalyst to detoxify acrylonitrile production wastewaters, it is necessary to significantly improve and enhance the stability of NHase. Stabilization of desired NHase activity was achieved over a broad range of thermal and pH conditions using simultaneous immobilization and chemical stabilization. Previously where 100% of NHase activity was lost in 24 hours in the non-stabilized cells, retention of 20% of initial activity was retained over 260 days when maintained at 50-55 C, and for over 570 days for selected catalyst formulations maintained at proposed temperature of the biodetoxification process. In addition, NHase and amidase enzymes from Rhodococcus sp. DAP 96253 were purified. Cell free NHase was characterized for its substrate range and effect of common enzyme inhibitors and was compared to available information for NHase from other organisms. As a result of this research a practical alternative to the deepwell injection of acrylonitrile production wastewaters is closer to reality.
6
Taylor, James Andrew. « The plasmids of Rhodococcus aetherivorans I24 ». Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613971.
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Oliveira, Luiz Gustavo Schneider. « Infecção por Rhodococcus equi em potros ». reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2013. http://hdl.handle.net/10183/75650.
Texte intégralRésumé :
Rhodococcus equi é um importante patógeno bacteriano em medicina veterinária, associado, sobretudo, a pneumonias piogranulomatosas em potros no primeiro semestre de vida. São descritos neste trabalho vinte casos de infecção por R. equi em potros recebidos para necropsia no Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul entre janeiro de 1997 e janeiro de 2013. Os históricos clínicos obtidos com os veterinários apresentaram grande variabilidade e, mesmo os sinais clássicos de comprometimento respiratório e febre só foram vistos em metade dos casos. Os dados obtidos em uma visita a uma propriedade demonstram que a superpopulação de potros e a introdução de fêmeas no grupo de parição contribuíram para a ocorrência de um surto. Exames de necropsia e de histologia revelaram que pneumonia piogranulomatosa multifocal foi a forma de apresentação mais constante (dezenove casos), seguida por linfadenite piogranulomatosa (dez casos) e tiflocolite piogranulomatosa e ulcerativa (cinco casos). Três animais apresentaram osteomielite piogranuloamatosa, dos quais, dois em vértebras. Uveítes e polissinovites assépticas foram constatadas em três casos. Exame imuno-histoquímco anti-Rhodococcus equi revelou-se positivo em todos os pulmões com lesões, embora os linfonodos tenham sido positivos em apenas três das nove amostras testadas. O exame bacteriológico das amostras de necropsia foi positivo em quinze casos clínicos, assim como em uma amostra de solo da propriedade visitada. O exame de reação da polimerase em cadeia (PCR) revelou o gene de virulência VapA de R. equi em todos os isolados clínicos, mas não na amostra de solo. Adicionalmente os pulmões foram testados por imuno-histoquímica para Pneumocystis sp.e apresentaram marcação em treze dos vinte casos.Rhodococcus equi is an important bacterial pathogen in veterinary medicine, especially associated with piogranulomatous pneumonia in foals under six months of age. Twenty cases of R. equi infection in foals received for necropsy at the Pathology Veterinary Sector (SPV) of the Federal University of Rio Grande do Sul (UFRGS) between January 1997 and January 2013 are described in this paper. Clinical history obtained with veterinary practitioners presented high variability, and even classical respiratory signs and fever were only observed in half of the cases. Data collected in an investigative visiting to a breeding farm showed that the foal superpopulation and the introduction of females to the parturition group contributed to the occurrence of an outbreak. Necropsy and histologic examinations revealed that multifocal piogranulomatous pneumonia was the most constant presentation (nineteen cases), followed by piogranulomatous lymphadenitis (ten cases) and piogranulomatous and ulcerative typhlocolitis (five cases). Three animals presented piogranulomatous osteomyelitis, two of them in vertebrae. Aseptic uveitis and polisynovitis were verified in three cases. Anti-Rhodococcus equi immunohistochemical examination stained positive in all lungs containing lesions, although lymphnodes have stained positive in only three of nine samples tested. Bacteriologic examination of the necropsy samples was positive in fifteen cases and in a soil sample from the visited breeding farm. Polymerase chain reaction (PCR) test revealed the VapA virulence factor of R.equi in all clinical isolates, but not in the soil sample. Additionally, the lungs were tested to the presence of Pneumocystis sp. by immunohistochemistry, and stained positive in thirteen of twenty cases.
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Chong, Chun Shiong. « Biodegradation of RDX in Rhodococcus spp ». Thesis, University of York, 2011. http://etheses.whiterose.ac.uk/1668/.
Texte intégralRésumé :
The manufacture, use and storage of explosives over decades have seriously contaminated the environment. Hexa-hydro-1,3,5-trinitro-1,3,5-triazine (Royal Demolition Explosive - RDX) is one of the most widely used explosives. RDX is a man-made compound, recalcitrant to degradation and proven to be toxic to organisms. Bacteria capable of utilising RDX as a sole nitrogen source for growth have been isolated from RDX polluted sites including Rhodococcus rhodochrous 11Y. The RDX degrading genes, xplA and xplB encoding a novel flavodoxin fused cytochrome P450 and its reductase partner respectively, were first identified in R. rhodochrous 11Y. This unusual P450 system has now been found in almost all of the RDX degrading bacteria isolated so far. To date, XplA/B remains the only characterised RDX degrading P450 system and is encoded on an operon in strain 11Y, which also contains a putative permease (transporter) and transcriptional regulator. This gene cluster is highly conserved amongst other RDX degrading bacteria from geographically distinct regions including the United Kingdom, Belgium, Australia and North America, suggesting the xplA/B gene cluster may have been rapidly distributed across the globe by horizontal gene transfer. The first aim of this study was to characterise Rhodococcus erythropolis HS4, a bacterium that degrades RDX slowly but did not contain xplA and xplB. A number of approaches have been employed to characterise this strain, which includes whole cell assays, western analysis, cell free extract activity assays, PCR amplification and affinity purification of protein in R. erythropolis HS4. While whole cells of HS4 showed low RDX degrading activity, no RDX activity was observed in HS4 cell free extracts. Attempts to purify an RDX degrading enzyme from R. erythropolis HS4 were unsuccessful. The second aim of this project was to characterise the RDX degrading gene cluster in R. rhodochrous 11Y. Four genes encoding xplB, xplA, a putative permease and a MarR type regulator, were individually deleted using an unmarked gene deletion system (pK18mobsacB). The xplB knockout strain metabolised RDX more slowly than the wild-type suggesting XplA was able to obtain reducing equivalents from another source in the xplB knockout strain. The xplA knockout strain did not show RDX activity in whole cell and growth experiments. Neither xplA nor its gene product XplA was detected in the xplA knockout strain. The findings suggested no alternative RDX degrading system is present in strain 11Y. The permease knockout did not show significant difference in the RDX removal rate compared to wild-type. Another attempt to characterise the permease in 11Y was to express it in E. coli. No significant difference of RDX uptake between the permease-expressing clone and the control (non-expressing clone) in the uptake assays. The regulator knockout strain had a lower RDX degradation rate than wild-type in the presence of nitrate/nitrite. Also, when the regulator knockout and wild-type strains were previously exposed to nitrate/nitrite, the knockout showed lower RDX degrading activity. Further investigation of xplA regulation in strain 11Y showed that XplA activity was induced by nitrogen-limiting conditions and further induced by RDX. This was the first observation on the XplA activity could be induced by low nitrogen availability in medium. These results suggested that the regulator is indirectly involved in controlling the expression of XplA in 11Y, which is linked to central nitrogen metabolism.
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Hunt, Jonathan Ralph. « Biotransformation of alkenes by Rhodococcus OU ». Thesis, University of Warwick, 1991. http://wrap.warwick.ac.uk/109487/.
Texte intégralRésumé :
Epoxides are an important class of synthons, produced in large quantities (notably epoxyethane and epoxypropane) for the manufacture of polymers. Reaction of epoxides with nucleophiles is stereospecific, offering a route to homochiral pharmaceuticals and agrochemicals from homochiral epoxides. With few exceptions, production of homochiral epoxides is difficult to achieve by chemical syntheses alone. However, alkene epoxidation by monooxygenase enzymes has been shown to proceed with a high degree of stereoselectivity in many instances. The aims of this project were to isolate microorganisms capable of converting alkenes to epoxides and to select the most suitable isolate for further characterization. Two Gram positive bacteria were isolated using α-methylstyrene (αMeS-1) and octane (Rhodococcus OU). The latter isolate was subjected to a more detailed study. Rhodococcus OU were shown to convert a range of structurally diverse alkenes to their corresponding epoxides: aliphatic (1-alkenes from propene to 1-tetradecene and cis-2- butene), alicyclic (cyclopentene and cyclohexene) and aromatic (styrene, allylbenzene and allylphenylether) alkenes. Alcohols, aldehydes and ketones were produced from alkenes with sub-terminal double bonds, in addition to epoxides. The stereoselectivity of alkene epoxidation was investigated by chiral HPLC. Partial resolution of (±)-1,2-epoxy-3-phenoxypropane was achieved, although assignment of the two peaks was not possible. Biotransformation of allyl phenyl ether to 1,2-epoxy-3- phenoxypropane was shown to proceed in a stereoselective manner. Problems associated with the chiral analysis of styrene oxide were not overcome, but preliminary results suggest that Rhodococcus OU is completely stereoselective for (R)-(+)-styrene oxide. Alkene epoxidation was shown to occur by one or more monooxygenase enzymes, expression of which is inducible by growth on n-alkanes but not by growth on 1-hexanol or glucose. Catalytic activity was retained after freezing in liquid nitrogen and storage at -70°C, only diminishing after being stored in excess of two months. Optimization of 1-alkene epoxidation was investigated, with particular reference to 1-hexene epoxidation. The specific rate of 1-alkene epoxidation (qp) was shown to increase as chain length decreased, correlating with an increase in 1-alkene solubility in water. Increasing the biocatalyst concentration resulted in an increase in volumetric productivity, but a decrease in qp. Epoxidation of 1-hexene showed saturable kinetics, qp being maximal between 0.05% to 0.10% (v/v) 1-hexene, whilst the final concentration of 1,2-epoxyhexane attained was concentration-dependant up to 0.40% (v/v) 1-hexene (the maximum concentration tested). Addition of co-substrates was not shown to enhance qp.
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Hullmann, Axel-Günther. « Prophylaxe der Rhodococcus-equi-Pneumonie bei Fohlen durch Vakzination mit Rhodococcus-equi-Impfstoff und Adjuvans CpG XXXX ». [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=984730109.
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Drago, Gene K. « Studies Directed to the Optimization of Fermentation of Rhodococcus sp. DAP 96253 and Rhodococcus rhodochrous DAP 96622 ». Digital Archive @ GSU, 2007. http://digitalarchive.gsu.edu/biology_diss/24.
Texte intégralRésumé :
Studies Directed to the Optimization of Fermentation of Rhodococcus sp. DAP 96253 and Rhodococcus rhodochrous DAP 96622 by GENE KIRK DRAGO Under the Direction of George E. Pierce ABSTRACT Bench- and pilot plant scale fed-batch fermentations were performed in stirred-tank bioreactors (STBR) with Rhodococcus sp. DAP 96253 and R. rhodochrous DAP 96622 in an attempt to elucidate parameters that may affect the optimization of a fermentation process for high biomass production and high inducible expression of cobalt-high-molecular-mass nitrile hydratase (Co-H-NHase. The effects of these factors on amidase (AMDase) activity were also investigated. Biomass and NHase production were inhibited by a total addition of acetonitrile and acrylonitrile (AC / AN) at 500 ppm during a 48 h run. Biomass and enzyme activity were uncoupled when the inoculum mass was increased from 4 g (wet weight) to ¡Ý 19 g. Other factors that allowed for the uncoupling of biomass production from enzyme activity were the reduction of the AC / AN feed rate from a step-addition at 2500 ¦Ìl / min to a continuous addition at 80 ¨C 120 ¦Ìl / min, and the delay to 18 h post-inoculation the time of initial inducer addition. The inhibition of both biomass production and NHase activity was relieved when both the total concentration of AC / AN was reduced to ¡Ü 350 ppm and the AC / AN feedrate was reduced. The factors with the greatest influence were shown to be the inducer, the inducer concentration, inoculum mass and source as well as the major carbohydrate and nitrogen source. In addition, this lab is the first to report high AN-specific NHase induction by asparagine (1300 ppm) in a fed-batch fermentation system. Prior to this program, 250 mg of cells (wet weight) per liter could be provided in 4 ¨C 10 days with an activity of 1 U NHase per mg of cells (dry weight). Current production is > 50 g / L in 48 h with an NHase activity > 150 U / mg of dry cell weight. INDEX WORDS: Amidase, Asparagine, Biodetoxification, Fermentation, Nitrile, Nitrile Hydratase, Rhodococcus
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Montoya, Crystal Leslie. « Opsonization of Rhodococcus equi decreases cytotoxic effects and modulates cytokine expression in equine macrophages ». Pullman, Wash. : Washington State University, 2010. http://www.dissertations.wsu.edu/Thesis/Spring2010/c_montoya_042610.pdf.
Texte intégralRésumé :
Thesis (M.S. in veterinary science)--Washington State University, May 2010.Title from PDF title page (viewed on July 22, 2010). "College of Veterinary Medicine." Includes bibliographical references (p. 26-35).
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Dang, Phuong-Nga. « Determining the functions of transcriptional regulatory genes of the npd gene cluster encoding 2,4,6-trinitrophenol degradation in Rhodococcus opacus HL PM-1 ». [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972630740.
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Kutanovas, Simonas. « Tetrametilpirazino skaidymo Rhodococcus sp. TMP1 bakterijose tyrimas ». Doctoral thesis, Lithuanian Academic Libraries Network (LABT), 2013. http://vddb.laba.lt/obj/LT-eLABa-0001:E.02~2013~D_20130701_092234-05318.
Texte intégralRésumé :
Alkilpirazinų katabolizmas bakterijose yra prastai ištirtas. Nors yra žinomi tarpiniai metabolitai susidarantys skaidant di- ir tri- pakeistus alkilpirazinus, tačiau šių junginių skaidymas prasideda hidroksilinimo reakcija, kuri negalima tetrametilpirazino atveju. Šiame darbe pirmą kartą nustatytas tetrametilpirazino katabolizmo kelias šį junginį skaidančiose Rhodococcus jostii TMP1 bakterijose. MS/MS de novo sekoskaitos būdu identifikavus tetrametilpirazinu indukuojamą baltymą, buvo nustatyta genų sankaupa, koduojanti pradines tetrametilpirazino katabolizmo reakcijas katalizuojančius fermentus ir transkripcijos reguliatorių, dalyvaujantį šių genų aktyvavime. Pradiniame tetrametilpirazino skaidymo etape monooksigenazė TpdAB katalizuoja oksidacinį žiedo atidarymą, susidarant (Z)-N,N'-(but-2-ene-2,3-diil)diacetamidui. Tolesnę skaidymo reakciją katalizuoja amidazė TpdC, kurios produktą N-(3-oksobutan-2-il)acetamidą aminoalkoholių dehidrogenazė TpdE redukuoja iki N-(3-hidroksibutan-2-il)acetamido. Nustačius tarpinius tetrametilpirazino skaidymo metabolitus, reakcijas katalizuojančius fermentus ir juos koduojančius genus buvo rekonstruotas pirmasis alkilpirazinų katabolizmo kelias bakterijose. Darbo metu taip pat parodyta, kad Rhodococcus jostii TMP1 bakterijos modifikuoja daugelį alkilpirazino ir alkilpiridino junginių ir gali būti panaudotos 2,4,6-trimetilpiridin-3-olio biosintezei iš 2,4,6-trimetilpiridino.The catabolism of alkylpyrazines is poorly described. The pathways for the degradation of di- and tri-substituted pyrazines have been proposed, but these related routes consistently include a hydroxylation step that cannot be performed on tetramethylpyrazine. Here we describe for the first time the catabolic pathway of tetramethylpyrazine in tetramethylpyrazine-degrading Rhodococcus jostii TMP1 strain. MS/MS analysis of the protein primarily upregulated by tetramethylpyrazine led to the identification of the gene locus encoding proteins required for the initial steps of tetrametylpyrazine degradation and for the regulation of this locus. Tetramethylpyrazine degradation starts with oxidative ring cleavage catalysed by monooxygenase TpdAB, which produces (Z)-N,N'-(but-2-ene-2,3-diyl)diacetamide. This compound is further hydrolysed by amidase TpdC to N-(3-oxobutan-2-yl)acetamide. TpdE was confirmed to be an aminoalcohol dehydrogenase yielding N-(3-hydroxybutan-2-yl)acetamide. By determining intermediates, enzymes involved and genes responsible for tetramethylpyrazine degradation we provide the first validated pathway for pyrazine degradation. We also report that Rhodococcus jostii TMP1 is capable of modifying various alkylpyrazines and alkylpyridines and can be employed for the bioconversion of 2,4,6-trimethylpyridine and 2,4,6-trimethylpyridin-3-ol biosynthesis.
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Fernandes, A. « Genetic tools for gene disruption in Rhodococcus ». Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598990.
Texte intégralRésumé :
The genetic analysis of the soil actinomycete Rhodococcus has been hampered by a lack of genetic tools. In recent years methods for gene cloning by gain of function into an E. coli or Rhodococcus host have been established. Methods for cloning Rhodococcus genes (particularly into E. coli) are fraught with difficulties, due to restriction/methylation of DNA, integration and ineffectual gene expression in the host. The establishment of a gene disruption system would overcome these difficulties and allow selection of useful phenotypes by loss of function. In this work a recently developed in vitro Tn5-based mutagenesis system was adapted for use of Rhodococcus. Electroporation protocols generating sufficient numbers of transformants were established and a random knockout library was constructed in a Rhodococcus type-strain. Part of this work involved investigations of Rhodococcus cell envelope ultrastructure and the use of growth supplements to aid transformation. Library coverage was investigated by the identification and sequencing of a number of amino acid auxotrophs. The Tn5-based system was applied to a wild-type soil Rhodococcus isolate and a random knockout library was constructed. A number of mutants unable to grow in the presence of toluene and benzene were isolated. A number of transposon delivery vectors based on either Tn5 or IS903 were constructed and problems of transposant selection overcome. For the purposes of construction the sequencing and analysis of two Rhodococcus plasmid replicons was carried out. The IS903-based vector although fully functional in E. coli failed to transpose in Rhodococcus and the possible reasons are discussed. Preliminary characterisation of a putative inducible promoter from Rhodococcus was carried out and the use of reporter genes yfp and luxAB established. The replicative Tn5 delivery vector was adapted to include the promoter/regulator to drive transposase expression however this vector was subjected to deletion in the Rhodococcus host.
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Santos, Valesca Peter dos. « Isolamento de bacteriófagos líticos para Rhodococcus sp ». reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2016. http://hdl.handle.net/10183/171045.
Texte intégralRésumé :
O Rhodococcus equi é uma das principais causas de pneumonia piogranulomatosa e linfadenite em potros de 1-6 meses de idade, resultando em alta morbidade e muitas vezes em óbito. Em propriedades endêmicas, a enfermidade é responsável por gerar altos custos para tratar e prevenir a doença. Com o objetivo de propor uma alternativa para o controle ambiental do Rhodococcus equi, foi aplicado um protocolo para isolamento de bacteriófagos. Foram isoladas 14 amostras de bacteriófagos, obtidas do ambiente de criação de cavalos. Estas apresentaram capacidade de lisar in vitro amostras de campo de Rhodococcus equi com diferentes intensidades. O resultado obtido nos possibilita sugerir que os fagos podem ser uma alternativa natural para a redução da bactéria no ambiente.Rhodococcus equi is an important cause of pyogranulomatus pneumonia and lymphadenitis in foals 1-6 months of age, resulting in high morbidity and often mortality. In endemic properties, the disease is responsible for generating high costs to treat and prevent disease. In order to propose an alternative to the environmental control of Rhodococcus equi, a protocol for isolation of bacteriophages was applied. Fourteen bacteriophage samples were isolated obtained from horse breeding environment. They showed capacity to lyse in vitro Rhodococcus equi field samples with different sensitivities. The results obtained allows us to suggest the phage as a natural alternative to reducing R. equi population.
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Kapadia, Jaimin Maheshbhai. « DNA transfer in the soil bacterium Rhodococcus ». Digital Commons @ East Tennessee State University, 2020. https://dc.etsu.edu/honors/565.
Texte intégralRésumé :
Gene transfer plays an important role in bacterial evolution. Especially in an under explored species like Rhodococcus, a type of bacteria found in the soil. Rhodococcus has several applications in the pharmaceutical industry and in the production of antibiotics. Rhodococcus possess several unique sets of properties which makes it beneficial to have a reliable method of producing mutants of Rhodococcus. The goal of the experiment was to find an efficient way of forming Rhodococcus colonies with kanamycin resistant genes. The project began from an unexpected observation from an earlier experiment with Rhodococcus strain MTM3W5.2. where I attempted to transform this strain with a transposon via electro-transformation. The colonies that grew/ appeared transformants were screened to confirm the presence of kanamycin gene, however there was no amplified DNA seen on the PCR gel (i.e. absence of the kanamycin gene). The electro-transformant colonies were selected on LB plates containing different higher concentrations of kanamycin. Then the appeared transformants were again screened via disk diffusion assay and were classified into 3 different kanamycin resistant phenotypes. Majority of the “C” phenotypic colonies (i.e., high level resistance to kanamycin) appear to contain the kanamycin gene, but these colonies were less in numbers. This led us to try another method of gene transfer which is conjugation. Conjugation was carried on a double selection antibiotic plate containing both chloramphenicol (30 µg) and kanamycin (100 µg). The transconjugate colonies that appeared on the double selection plates were also screened by PCR, but none of the colonies had amplified DNA suggesting absence of the kanamycin gene. The colonies seen on the double selection plate were possibly due to spontaneous mutation or some type of unknown phenotypic variation. However, in the future, double selection plates with higher concentrations of antibiotics can possibly give us transconjugants with kanamycin genes.
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Meka-apiruk, Junpen. « Carotenoid biosynthesis in Rhodococcus maris strain N1020 ». Thesis, University of Liverpool, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321160.
Texte intégral
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Morais, Amanda Bonalume Cordeiro de. « Ocorrência de patógenos de origem bacteriana e viral e marcadores de virulência de Escherichia coli e Rhodococcus equi isolados das fezes de aves silvestres de cativiero da fauna brasileira / ». Botucatu, 2014. http://hdl.handle.net/11449/108774.
Texte intégralRésumé :
Orientador: Márcio Garcia RibeiroBanca: Carlos Roberto Teixeira
Banca: Jean Carlos Ramos da Silva
Resumo: O presente estudo investigou a ocorrência de Escherichia coli, Rhodococcus equi, Salmonella sp., Coronavírus e Rotavírus nas fezes de Passeriformes e Psitaciformes pertencentes à fauna nacional, de 29 diferentes espécies, sem sinais entéricos. Foram investigados também marcadores de virulência nas linhagens de E. coli (cnf1, hly, papC, papGI, papGII, papGIII, fimH, afa, sfa, iucD, usp, vt1, vt2, eae, k88) e R. equi (genes vapA e vapB). As aves utilizadas no estudo foram provenientes do Centro de Medicina e Pesquisa em Animais Silvestres (CEMPAS) FMVZ - UNESP/ Botucatu, SP, do Parque Zoológico Municipal "Quinzinho de Barros" (PZMQB) de Sorocaba, SP e de criadores particulares com aves registradas no Instituto Brasileiro do Meio Ambiente e dos Recursos Renováveis (IBAMA) da região de Botucatu, SP. Do total de 152 amostras avaliadas foram isoladas 46 (30,26%) linhagens de E. coli das quais 37 (80%) foram provenientes de amostras de Psitaciformes e 9 (20%) de Passeriformes. Houve diferença significante (p<0,05) entre os grupos para o maior isolamento de E. coli nos Psitaciformes. Dentre os marcadores de virulência de E. coli foram detectados os genes fim H (58,69%) e eae (4,34%). Foram isoladas 2 (1,32%) linhagens de R. equi, todas de Psitaciformes. Nestes isolados de R. equi não foram identificados os genes vapA e vapB associados à virulência. Foi encontrado material genético de Rotavírus bovino em três (1,97%) amostras de Psitaciformes. Salmonella sp. e Coronavírus não foram identificados nas aves amostradas. A presença de E. coli, R. equi e Rotavírus em amostras de fezes de aves silvestres, sem sinais entéricos, reforça o potencial destas espécies de servirem como reservatórios de patógenos de eliminação entérica para os humanos, devido à presença destes animais no ambiente domiciliar e peridomiciliar
Abstract: The present study investigated the occurrence of Escherichia coli, Rhodococcus equi, Salmonella sp., Coronavirus and Rotavirus in the feces of Passeriformes and psittaciformes belonging to Brazilian wildlife, from 29 different species, without enteric signs. Virulence markers were also investigated in strains of E. coli (cnf1, hlyA, papC, papGI, papGII, papGIII, fimH, afa, sfa, iucD, usp, vt1, vt2, eae, k88) and R. equi (vapA and vapB genes). The birds used in the study came from the Centro de Medicina e Pesquisa em Animais Silvestres (CEMPAS) FMVZ - UNESP / Botucatu, SP, Parque Zoológico Municipal "Quinzinho de Barros" (PZMQB) Sorocaba, SP and private breeders with birds recorded in Instituto Brasileiro do Meio Ambiente e dos Recursos Renováveis (IBAMA) from Botucatu region, SP. Of the total 152 fecal samples evaluated were isolated 46 (30.26%) strains of E. coli. From these, 37 (80%) were from psittaciformes samples and 9 (20%) of Passeriformes. There was a statistical difference (p <0.05) between groups with greater isolation of E. coli in psittaciformes. Among the virulence markers of E. coli were detected the genes fimH (58,69%) and eae (4,34%). Were isolated 2 (1.32%) R. equi strains, all from psittaciformes. Among these R. equi isolates any vapA and vapB genes associated with virulence were founded. Genetic material of bovine Rotavirus was found in three (1.97%) psittaciformes samples. Salmonella sp. and Coronavírus weren't identified in any of the sampled birds. The presence of E. coli, R. equi and Rotavirus in fecal samples of wild birds without enteric signs from Brazil wildlife, reinforces the potential of these birds as a reservoirs of pathogens of enteric elimination for humans, due to the presence of these animals in the domestic and peridomestic, environment of human
Mestre
20
Miclo, André. « Catabolisme de l'androst-4-ène-3,17-dione par Rhodococcus equi ». Vandoeuvre-les-Nancy, INPL, 1988. http://www.theses.fr/1988NAN10295.
Texte intégral
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Kreit, Joseph. « Catabolisme microbien des stérols : caractérisation de la cholestérol oxydase, de la cholestérol ester hydrolase et d'une alcool secondaire deshydrogénase chez Rhodococcus Sp. CIP 105 335 ». Vandoeuvre-les-Nancy, INPL, 1999. http://www.theses.fr/1999INPL044N.
Texte intégralRésumé :
Revue Le catabolisme microbien des stérols passe par deux voies distinctes. L'une concerne la dégradation du noyau stéroïde, tandis que l'autre concerne le clivage de la chaîne latérale. Ce clivage est effectué par oxydation. Les actinomycètes sont généralement actifs dans le catabolisme des stérols. Le clivage sélectif de la chaîne latérale des stérols, qui fournit le noyau stéroïde est d'un intérêt économique remarquable. La cholestérol oxydase (COX), première enzyme du catabolisme des stérols, transforme la structure 5-ène-3[bêta]-ol en 4-ène-3-one. Les COXs microbiennes sont produites sous forme sécrétée et/ou sous forme cellulaire. Elles sont monomériques (54-62 kDa) et polymorphes. Le gène de l'enzyme et son opéron ont été identifiés chez une espèce de Streptomyces. Ce gène a été aussi étudié chez Brevibacterium sterolicum. Les COXs sont utilisées dans plusieurs applications. Resultats Deux isolats du sol (GK1, GK3), actifs dans le catabolisme des stérols, ont été identifiés comme appartenant au genre Rhodococcus. La souche GK1 (CIP 105335) produit la COX sous formes extracellulaire et cellulaire. La COX cellulaire est localisée à l'extérieur de la membrane cytoplasmique. Un essai calorimétrique a été mis au point pour doser l'activité de la COX. Il permet de doser l'enzyme acellulaire, et aussi l'enzyme liée aux cellules sans qu'elle soit extraite. L'induction de la COX de Rhodococcus sp. GK1 est liée à la chaîne latérale des stérols. La structure 3[bêta]-ol-5-ène ne suffit pas pour l'induire. La culture de cette souche sur stérols, comme seules sources de carbone et d'énergie, est marquée par la production de la COX cellulaire uniquement. Cette forme est extractible par traitement des cellules avec des détergents non ioniques. La croissance de GKl sur une source de carbone différente des stérols, ou sur un stérol plus un autre composé carboné, est marquée par une sécrétion importante de la COX. Les formes de la COX de GK1 ont la même spécificité de substrats. La double liaison en C-5 n'est pas nécessaire pour l'oxydation de la fonction alcool. La longueur de la chaîne latérale des stérols influence le niveau de l'activité. Ces formes possèdent un certain caractère hydrophobe. Le Triton X-100 ou le Lubrol PX (0,1- 0,2 %, p/v) stimulent leur activité. La COX de GK1 est relativement stable à des températures allant jusqu'à 50°C. La COX extracellulaire et cellulaire de cette souche ont été purifiées. Leur masse moléculaire est environ 59 kDa. La séquence en aminoacides du ségment N-terminal de la COX extracellulaire est: H2N-Ala-Pro-Pro-Val-Ala-Ser-XArg- Tyr-X-(Phe)-. Le pl de cette forme est de l'ordre de 9,0. Une cholestérol ester hydrolase (estérase) a été mise en évidence dans les cellules de GKl. Une alcool secondaire déshydrogénase NAD-dépendante a été obtenue de cette souche et caractérisée. Sa masse moléculaire apparente est 60 kDa. Elle catalyse la réduction des monocétones et des dicétones comme le 2-pentanone et le diacétyle. Elle oxyde des alcools secondaires comme l'isopropanol et le 2-octanol.
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Nagarajan, Jayasudha. « Metabolism of halogenated compounds by Rhodococcus UKMP-5M ». Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/34383.
Texte intégralRésumé :
Members of the genus Rhodococcus are well known for their high metabolic capabilities to degrade wide range of organic compounds ranging from simple hydrocarbons to more recalcitrant compounds such as polychlorinated biphenyls. Their ability to display novel enzymatic capabilities for the transformation of many hazardous contaminants in the environment makes them a potential candidate for bioremediation. Rhodococcus UKMP-5M, an actinomycete isolated in Peninsular Malaysia shows great potential towards degradation of cyanide, hydrocarbons and phenolic compounds. In the present study, the capacity of this strain to degrade halogenated compounds was explored. Preliminary investigations have proven that R. UKMP-5M was not able to utilise any of the halogenated compounds tested as sole carbon and energy source, but the resting cells of R. UKMP-5M was able to dechlorinate several compounds which include chloroalkanes, chloroalcohols and chloroacids and the activity was three fold higher when the cells were grown in the presence of 1-Chlorobutane (1-CB). Therefore, 1-CB was chosen as a substrate to unravel the mechanism of dehalogenation in R. UKMP-5M. In contrast to the classic hydrolytic route for the assimilation of 1-CB in many organisms, R. UKMP-5M was able to metabolise and release chloride from 1-CB, but is unable to use the product from 1-CB metabolism as growth substrate. On comparing the protein profiles of the induced and non-induced cells of R. UKMP-5M, two types of monooxygenases were identified in the induced condition, which were not present in the uninduced sample. The strict oxygen requirement for dechlorination of 1-CB and the identification of monooxygenases in the induced protein extract suggests that 1-CB dehalogenation is likely to be catalysed by a monooxygenase. In addition to these monooxygenases, a protein that was later identified as amidohydrolase (Ah) was also found to be induced when the cells were exposed to 1-CB. Therefore, Ah from R. UKMP-5M was cloned and expressed in E. coli to test the ability of the purified Ah to release chloride from 1-CB. The heterologous expression of Ah in E. coli resulted in the formation of inclusion bodies and the western blot analyses further confirmed that no soluble form of Ah was present. Multiple attempts to obtain a soluble and functionally active Ah were not successful. Therefore, on-column refolding was carried out to obtain a biologically active Ah. A 3D model based on structural homology was predicted as a preliminary step to characterize this protein. However, when assayed with 1-CB, Ah was found not to catalyze dehalogenation. All results of this thesis suggest that metabolism of 1-CB by R. UKMP-5M is via γ-butyrolactone which acts as a potent intracellular electrophile that covalently modifies proteins and nucleic acids. The findings from this research are important to determine the metabolic capacity of a Malaysian Rhodoccoccus in dehalogenation of halogenated compounds and its potential application in bioremediation.
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Diaz, Salazar Albelda Carlos. « Redundancy in the biosynthesis of triacylglycerol by Rhodococcus ». Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/58982.
Texte intégralRésumé :
Many mycolic acid-containing actinobacteria are oleaginous, accumulating high amounts of triacylglycerols (TAGs) under conditions of nutrient stress. These bacteria contain multiple copies of the genes involved in TAG biosynthesis: glycerol-phosphate acyltransferase (GPAT), acylglycerol-phosphate acyltransferase (AGPAT), phosphatidic acid phosphatase (PAP) and diglyceride acyltransferases (WS/DGAT), encoded by plsB, plsC, pap, and atf, respectively. Analysis of Rhodococcus jostii RHA1’s genome revealed that it carries 1 plsB, 8 plsC, 7 pap, and 16 atf. Quantitative, time-dependent data of six of these atf genes, selected based on previous transcriptomics data, revealed distinct expression patterns under nitrogen-limiting (N-) and carbon-limiting (C-) conditions. For example, the levels of atf10, atf3 and atf8 transcripts dropped ~10-fold upon growth substrate depletion, while the levels of atf4, atf6 and atf9 transcripts rose. Under N- conditions, RHA1 cells continued to accumulate TAGs for five days after ammonia depletion, during which time atf10 and atf8 transcripts remained abundant. Targeted deletion of any one of atf3, atf4, atf6, atf9 and atf10 did not significantly affect TAG accumulation under N- conditions, consistent with the redundancy of putative acyltransferases in the RHA1 genome. However, deletion of both atf8 and atf10 resulted in a 50% decrease in TAG accumulation. Furthermore, the fatty acid profile of the ∆atf8∆atf10 mutant was significantly perturbed, and was restored by complementation with either atf8 or atf10. RT-qPCR data analysis also revealed that the expression patterns of plsC (RS27555) and plsB were the same as that of atf9, consistent with their occurrence in an operon. Unexpectedly, deletion of plsB did not affect TAG accumulation, suggesting an alternative pathway for TAG and phospholipids biosynthesis. Finally, I identified three genes encoding HAD-type hydrolases as being putatively involved in TAG biosynthesis, including one that occurs as a fusion with plsC. The available data suggest that they act as PAPs. Overall, the results establish that there is a certain degree of functional redundancy in TAG biosynthesis, and that Atf8 and Atf10 play a major role in TAG accumulation. At the same time, the results also highlight important gaps in our knowledge of TAG biosynthesis in mycolic acid-containing oleaginous actinobacteria.Science, Faculty of
Graduate
24
Dhandapani, Pragathi Dhandapani. « Rhodococcus fascians-plant interactions : microbiological and molecular aspects ». Thesis, University of Canterbury. Biological Sciences, 2014. http://hdl.handle.net/10092/9281.
Texte intégralRésumé :
Rhodococcus fascians, a plant pathogenic actinomycete with a very broad host range, causes leafy galls and other malformations. The plant hormone, group, the cytokinins has been implicated in the alteration of host morphology. The aim of this project was to gain insight into the interaction of the cytokinin biosynthetic, isopentenyltransferase (IPT), cytokinin activating ( LOG (The Lonely Guy)) and the cytokinin metabolic, cytokinin oxidase/dehydrogenase (CKX) gene families of both Pisum sativum and R. fascians during infection of the plant.
R. fascians colonisation and infection of pea were examined using scanning electron microscopy (SEM) and light microscopy. The expression of genes related to cytokinin biosynthesis, activation and metabolism were isolated and assessed with polymerase chain reaction (PCR) and real time-quantitative PCR (RT-qPCR) analysis. Primers were designed to discriminate between pea genes and R. fascians genes. In addition, the response of the pea cotyledons to R. fascians was measured through chlorophyll estimation and the expression of the transporter genes, sucrose transporter (SUT) and amino acid permease (AAP) which were assayed through RT-qPCR. The pea response regulators were monitored as an indirect measure of the level of endogenous cytokinins in pea.
Two R. fascians strains, the avirulent strain 589 and the virulent strain 602, were selected for this project based on their virulence and similar growth rate under identical conditions. The virulence of R. fascians virulent strain 602 was also confirmed through Koch's postulates. The phenotypic alterations in the pea infected with the virulent strain 602 included stunted growth, multiple shoots, small leaves, thickened primary roots and reduced secondary root growth. Delayed senescence of shoots and dark green, intact cotyledons were also observed. Microscopic analyses revealed epiphytic colonisation by both the avirulent strain 589 and the virulent strain 602 in pea cotyledons, roots, shoots and leaves and endophytic colonisation in the seed coat from the time of seed inoculation to 45 days post inoculation (dpi).
The expression of R. fascians genes was relatively high at 5 and 9 dpi in pea cotyledons and at 15 and 25 dpi in roots and shoots of pea infected with the virulent strain 602. The expression of RfIPT, RfLOG and RfCKX was not detected both in the control pea and the pea infected with the avirulent strain 589.
The cytokinin biosynthesis, metabolism and response regulator (RR) multi-gene families of PsIPTs, PsLOGs, PsCKXs and PsRRs revealed differential and tissue-specific expression patterns. The expression of PsIPTs and PsLOGs was induced immediately after inoculation with the R. fascians virulent strain 602 in the cotyledon but not in roots and shoots, and the expression level reduced at later growth stages. The PsCKXs and PsRRs expression level increased with the growth of the host infected with the virulent strain 602. In pea infected with the avirulent strain 589 the expression of PsIPTs, PsLOGs and PsCKXs gene family members generally increased after 25 dpi in cotyledons, roots and shoots, whereas PsRRs expression was low at all time points.
The up-regulation of PsIPTs and PsLOGs immediately after inoculation in cotyledons and at 15 dpi in roots and shoots by R. fascians virulent strain leads to elevated cytokinins which is reflected by the up-regulation of PsRRs. The plant responds to elevated cytokinin by producing phenotypic changes including shoot malformations. The plant activates its cytokinin homeostasis mechanism due to change in cytokinin level which is indicated by up-regulation of PsCKXs. Generally, the expression of the PsRRs was also up-regulated over time following infection by the R. fascians virulent strain. This indicates the presence of biologically active cytokinins in the host which maintain the symptoms. The outcome due to the avirulent strains indicates that, even though PsIPTs and PsLOGs are up-regulated at later growth stages (25 to 35 dpi), expression of PsCKX gene families were varied (either up-regulated or down regulated after 25 dpi). However, PsRRs expression was down-regulated suggesting low cytokinins levels in tissues which may be due to the activation of homeostatic mechanisms of the plant to reduce the level of biologically active cytokinins.
The chlorophyll content increased in cotyledons infected with the virulent strain 602 and PsSUTs and PsAAPs expression pattern in pea cotyledon and shoot infected with the virulent strain 602 indicates that R. fascians converts the infected tissue into a sink for their establishment and growth.
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Delcroix, Valerie A. « Aromatic compound degradation by cresol-utilizing Rhodococcus strains ». Thesis, Queen's University Belfast, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263367.
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Gradley, Michelle Lorraine. « Biotransformation of nitriles by Rhodococcus rhodocrous NCIMB 11216 ». Thesis, University of Kent, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240163.
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Strachan, Philip. « Catechol 1,2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 ». Thesis, University of Glasgow, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.337527.
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Jones, Amanda Louise. « Towards an improved taxonomy of the genus 'Rhodococcus' ». Thesis, University of Newcastle Upon Tyne, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.432583.
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OLIVERA, CARLOS ALBERTO CASTANEDA. « BIOFLOTATION OF HEMATITE USING THE BACTERIA : RHODOCOCCUS ERYTHROPOLIS ». PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO, 2014. http://www.maxwell.vrac.puc-rio.br/Busca_etds.php?strSecao=resultado&nrSeq=25131@1.
Texte intégralRésumé :
PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIROCONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO
A crescente demanda mundial por matérias-primas minerais levou ao aumento da exploração mineral e paralelamente novas pesquisas estão sendo dirigidas para a produção de novos reagentes de flotação, a fim de que estes apresentem maior seletividade e não sejam agressivos ao meio ambiente. Nesta pesquisa teve-se como objetivo estudar os aspectos fundamentais da bioflotação da hematita, avaliando a cepa bacteriana Rhodococcus erythropolis como biocoletor. Entre os estudos efetuados estão análises química para determinar as proteínas e carboidratos presentes no concentrado bacteriano, estabelecendo-se que é constituída por macromoléculas com características anfipáticas. O balanço entre grupos catiônicos e aniônicos da bactéria atribui um ponto isoelétrico (PIE) equivalente de 2,2. O perfil de potencial zeta da amostra mineral de hematita após interação com a bactéria mostrou uma mudança, onde o PIE mudou de 5,3 para 2,1. Para o estudo dos ensaios de adesão e microflotação, a amostra foi condicionada com a biomassa por meio de agitação sob condições específicas, tais como os tamanho de partícula, a concentração de biomassa, o pH da solução e tempo de condicionamento. A adesão da biomassa na superfície do mineral foi maior em pH 2 e na concentração de 200 mg / L. Os testes de microflotação foram feitos num tubo de Hallimond e foi avaliado a formação de espuma para uma concentração bacteriana de 200 mg / L, onde foi observado que a tensão superficial da solução aumenta à medida que o pH se torna básico. Das três faixas granulométricas utilizadas, a maior flotabilidade (83.86 por cento) foi alcançada na fração granulométrica (53 - 38 um), num pH 6 e com um tempo de flotação de 10 min. A bioflotação do mineral hematita segue o modelo cinético de segunda ordem, observou-se que as constantes de taxa (K2) da flotação do mineral aumentam com reduções de tamanho de partícula, mudando de 0,16369 (g.min)(-1) para 0,51604 (g.min)(-1) quando o tamanho de particula passou de (150 - 106 um) para (53 - 38 um). Os resultados apresentados mostram que o estudo do comportamento da cepa bacteriana Rhodococcus erythropolis como bioreagente na flotação de hematita foi viável, demonstrando o seu potencial uso como bioreagente coletor de mineral hematita, e assim projetando-se para uma futura aplicação na indústria da flotação mineral.
The growing world demand for raw minerals has led to the increased mineral exploration and at the same time new research is being directed toward the production of new flotation reagents, so that they present higher selectivity and they are environmentally friendly. This research aimed to study the fundamental aspects of bioflotation of hematite, evaluating the bacterial strain Rhodococcus erythropolis as biocollector. Among the studies are conducted chemical analyzes to determine the proteins and carbohydrates present in the bacterial concentrate, it was established that is composed of macromolecules with amphipathic characteristics. The balance between cationic and anionic groups of the bacteria assigns an equivalent isoelectric point (IEP) of 2.2. The profile of zeta potential of the sample of hematite mineral after interaction with the bacteria showed a change, where the IEP has changed from 5.3 to 2.1. To study the adhesion and microflotation assays, the mineral sample was conditioned with the biomass by stirring under specific conditions, such as the particle sizes, biomass concentration, the pH of the solution and conditioning time. The biomass adhesion on mineral surface was higher at pH 2 and at the concentration of 200 mg / L. The microflotation tests were carried out in Hallimond tube and was evaluated the foam formation to a bacterial concentration of 200 mg / L, which was observed that the surface tension of the solution increases as the pH becomes basic. Of the three granulometric fractions used, the greatest floatability (83.86 percent) was achieved in the granulometric fraction (53 - 38 um), at pH 6 and with a flotation time of 10 min. The hematite mineral bioflotation follows the second-order kinetic model, was observed rate constant (K2) of the mineral flotation increase with reductions of particle size, moving from 0,16369 (g.min)(-1) for 0,51604 (g.min)(-1) when the particle size changed from (150 - 106 um) to (53 - 38 um). The results presented show that the study of the behavior of the bacterial 10 strain Rhodococcus erythropolis as bioreagent in the flotation of hematite was feasible, demonstrating its potential use as collector bioreagent of mineral hematite, and so projecting into a future application in the mineral flotation industry.
30
Yang, Joyce Chun-Yi. « Conjugation of extrachromosomal replicons of Rhodococcus erythropolis AN12 ». Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/34575.
Texte intégralRésumé :
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2006.Includes bibliographical references.
Bacteria belonging to the Gram-positive actinomycete species, Rhodococcus erythropolis, are diverse not only in terms of metabolic potentials but the plasmids they encode. Pulsed-field gel electrophoresis (PFGE) revealed three previously uncharacterized megaplasmids in the genome of Rhodococcus erythropolis AN12. These megaplasmids, pREA400, pREA250 and pREA100, migrate at approximately 400 kb, 250 kb and 100 kb, respectively. Genetic screening of an AN12 transposon insertion library showed that two megaplasmids, pREA400 and pREA250, are conjugative. It is known for other bacterial systems that a relaxase encoded by the traA gene is required to initiate DNA transfer during plasmid conjugation. Sequences adjacent to the transposon insertion in megaplasmid pREA400 revealed a putative traA-like open reading frame. A novel site-specific gene disruption method was developed to generate a traA mutation in AN12, which allowed us to address the role of the traA gene for Rhodococcus megaplasmid conjugation. We found that the AN12 traA mutant is no longer capable of transferring the pREA400 megaplasmid to Rhodococcus erythropolis SQ1. It was shown previously that the R. erythropolis AN12 genome harbors a 6.3 kb cryptic plasmid called pAN12.
(cont.) Here we show that pAN12 is conjugatively mobilizable into other rhodococcal strains. A series of plasmid deletion constructs were tested for loss of mobility to identify the pAN12 cis-acting conjugation requirement. In this way, an approximately 700 bp region was found to be required for plasmid transmission. A small 61 bp element within this region exhibited sequence similarity to the minimal 54 bp clt region known to be required for the conjugation of the streptomycete plasmid, pIJ101. The functionality of these cis-acting elements appears to be conserved, as the addition of this pAN12 clt-like region confers mobility to an otherwise non-conjugative plasmid. However, unlike pJI 101 which encodes all necessary factors for transfer, pAN12 mobility is dependent on the presence of the AN12 megaplasmid, pREA400.
by Joyce Chun-Yi Yang.
Ph.D.
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Boccadoro, Catherine. « Biotransformation of 2,4,6-trinitrotoluene by novel Rhodococcus spp ». Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614035.
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Hapeshi, Alexia. « Chromosomal and plasmid determinants of Rhodococcus equi virulence ». Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/17281.
Texte intégralRésumé :
Rhodococcus equi is a soil-dwelling actinomycete with the ability to cause pyogranulomatous infections in different animal species and people. Young foals are particularly susceptible and develop a severe pulmonary illness known as rhodococcal pneumonia. The infection is endemic in many horse-breeding farms worldwide and poses a major challenge to the equine industry, as there is no commercial vaccine available. R. equi is a facultative intracellular pathogen. Intracellular survival in macrophages and hence virulence depends on the presence of large plasmids that carry a set of genes encoding virulence-associated proteins (Vaps) of largely unknown functions. Virulence plasmids are of different types and appear to determine host-specific infectivity for horses, pigs and cattle. In this thesis, I explored bacterial chromosomal factors that contribute to the virulence of R. equi. Previous microarray transcription profiling work from the laboratory showed that housekeeping metabolic genes from the R. equi chromosome were co-opted to serve a virulence function via co-regulation with plasmid virulence genes. Here, I identified a further virulence plasmid-co-expressed metabolic chromosomal locus with a key role in R. equi pathogenesis. The identified locus, gltAB1, encodes an NADPH-dependent glutamate synthase required for ammonia assimilation under low nitrogen conditions. Reverse-transcription quantitative rea-ltime PCR confirmed that gltAB1 was co-expressed with the vap genes from the plasmid whereas a homologous chromosomal locus encoding a second NADPH-dependent glutamate synthase, gltAB2, was not. In-frame deletion mutants were constructed and their virulence analysed. gltAB1 but not gltAB2, was found to be involved in virulence and required for intracellular proliferation in J774A.1 macrophages. The ΔgltAB1 mutant showed significant attenuation in vivo in a mouse infection model, in contrast to the ΔgltAB2, which behaved like the wild type. The ability of the ΔgltAB1 mutant strain to act as a live attenuated vaccine was tested in experiments in BALB/c mice. The mutant conferred protection against subsequent challenge of the animals with wild-type virulent bacteria, thus identifying a novel candidate vaccine for the control of R. equi pneumonia in foals. Furthermore, this thesis describes studies of the bovine-type plasmid, previously sequenced in our laboratory. The purpose of this work was to determine if VapN, the bovine-type allelic variant of the VapA protein encoded in the equine-type plasmid, was also essential for R. equi virulence. A plasmid-less derivative strain and a deletion mutant in the vapN gene were examined for their ability to proliferate in two different cell lines and to persist in BALB/c mice. These strains showed the same strong virulence attenuation observed with plasmid-less and ΔvapA strains derived from the equine isolate R.equi 103S, demonstrating that the bovine-type VapN protein also plays a central role in R. equi virulence. Additionally, the thesis includes preliminary work on approaches to explore the role and mechanism of Vap proteins in R. equi virulence. It also describes the construction of GFP-tagged R. equi strains for use in cell biological experiments and live imaging of infected cells.
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Forizs, Laetitia. « Metabolism and pathogenicity in the phytopathogen Rhodococcus fascians ». Doctoral thesis, Universite Libre de Bruxelles, 2012. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209742.
Texte intégralRésumé :
Rhodococcus fascians is a Gram-positive phytopathogenic bacterium which induces the development of leafy galls, local amplifications of multiple buds, on most infected plants. This process is linked to the production of phytohormones along with the presence of essential virulence-associated genes like the plasmid loci att and fas and the chromosomal gene vicA. However, the presence of these genes is not sufficient to ensure the infection phenotype development, indicating that other genes play a role in R. fascians pathogenicity. In this work, we studied the metabolic modifications occurring when the bacterium interacts with its host using a proteomic approach. A comparison between virulent and avirulent strains showed variations in the expression of catalases. In the virulent strain, besides the transitory induction of the att locus expression, the bacterium changes its metabolism from the Krebs cycle to the glyoxylate shunt, a process which is frequently observed in bacteria confronted to a hostile environment. The expression of the shunt-specific enzyme isocitrate lyase increased, while expression of fumarate hydratase and pyruvate dehydrogenase decreased. Hence, we focused on the link between the glyoxylate shunt and virulence. A screening of a R. fascians mutant library based on the capacity of bacteria to use acetate as the sole carbon source, a metabolic pathway depending on the glyoxylate shunt, resulted in the identification of a new gene essential for R. fascians pathogenicity. This gene encodes a glycosyl transferase, an enzyme known to be involved in the bacterial cell wall biosynthesis but possibly also implicated in cytokinin secretion. A mutant in this gene harboured an altered colony phenotype and could not induce malformations on infected plants. Accordingly, our results were integrated in the leafy gall pathology model recently presented by Stes et al. (2011). Finally, the several questions that are raised by this work, allowed us to suggest further research perspectives in order to unveil a little more of the R. fascians mysterious ways to interact with the plant./Rhodococcus fascians est une bactérie Gram-positive phytopathogène qui induit le développement de galles feuillées, des amplifications locales de multiples bourgeons, sur la plupart des plantes infectées. Ce processus est lié à la production de phytohormones ainsi qu’à la présence de gènes essentiels associés à la virulence tels que les loci plasmidiques att et fas et le gène chromosomique vicA. Cependant, la présence de ces gènes ne suffit pas à garantir le développement du phénotype d’infection, indiquant que d’autres gènes jouent un rôle dans la pathogénicité de R. fascians. Dans ce travail, nous avons étudié les modifications métaboliques qui se produisent lorsque la bactérie interagit avec son hôte par une approche protéomique. Une comparaison entre les souches virulente et avirulente a mis en évidence des variations d’expression au niveau des catalases. Dans la souche virulente, outre l’induction transitoire de l’expression du locus att, la bactérie change son métabolisme pour passer du cycle de Krebs au shunt du glyoxylate, un processus fréquemment observé chez les bactéries confrontées à un environnement hostile. L’expression de l’isocitrate lyase, enzyme spécifique au shunt, augmente, tandis que celle de la fumarate hydratase et de la pyruvate déhydrogénase diminue. Nous nous sommes donc intéressés au lien entre le shunt du glyoxylate et la virulence. Le screening d’une banque de mutants de R. fascians basé sur la capacité de la bactérie à utiliser l’acétate comme seule source de carbone, une voie métabolique dépendant du shunt du glyoxylate, a permis d’identifier un nouveau gène essentiel pour la pathogénicité de R. fascians. Ce gène code pour une glycosyl transferase, une enzyme impliquée dans la biosynthèse de la paroi bactérienne mais également dans la sécrétion des cytokinines. Un mutant dans ce gène présente un phénotype de colonie altéré et ne peut induire de malformations chez les plantes infectées. Finalement, nos résultats et les pistes d’interprétations que nous avons émisent nous permettent de compléter le modèle de l’interaction R. fascians-plante proposé récemment par Stes et al. (2011). Des perspectives de recherches visant une meilleure compréhension de ce pathosystème sont proposées.Doctorat en Sciences
info:eu-repo/semantics/nonPublished
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Sahin, Orhan. « Development of a Selective Medium for Rhodococcus Equi ». The Ohio State University, 1997. http://rave.ohiolink.edu/etdc/view?acc_num=osu1396440093.
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Conrad, Catleen. « Bernhard-von-Cotta-Preis 2014 : Reinigung und Charakterisierung von zwei Peroxidasen DypA und DypB aus Rhodococcus opacus 1CP ». Technische Universitaet Bergakademie Freiberg Universitaetsbibliothek "Georgius Agricola", 2016. http://nbn-resolving.de/urn:nbn:de:bsz:105-qucosa-211185.
Texte intégralRésumé :
Das Bodenbakterium Rhodococcus opacus 1CP ist für sein Potenzial bekannt, organische Schadstoffe abzubauen. In dieser Studie lag der Fokus auf zwei Genprodukten, den farbstoffabbauenden Peroxidasen DypA und DypB, welche unter Anwendung von Klonierungs- und Expressionsstrategien mit hohen spezifischen Aktivitäten in reiner Form gewonnen werden konnten. Eine umfassende biochemische Analyse zeigte, dass beide Enzyme in der Lage sind, Farbstoffe unterschiedlicher Klassen, insbesondere jedoch, die als schwer abbaubar geltenden Anthraquinone, strukturell anzugreifen. Damit stellen sie lohnenswerte enzymatische Systeme zum Abbau toxischer Farbstoffe beispielsweise in industriellen Abwässern dar.
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Santos, Inajara Silveira dos. « Actinomicetoses no Rio Grande do Sul : a propósito de 59 casos, atualizando actinomicose, nocardiose e rodococose ». reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2010. http://hdl.handle.net/10183/30943.
Texte intégralRésumé :
Descrição: As doenças causadas por actinomicetos patógenos, aeróbios e anaeróbios facultativos, diferem consideravelmente no que diz respeito à sua etiologia, patogênese, apresentação clínica e epidemiologia. Objetivos: Analisar a distribuição etária, manifestações clínicas, doenças de base e condições associadas, achados radiológicos, microbiológicos, tratamento e evolução, nos pacientes com actinomicetoses (actinomicose, nocardiose, rodococose). Delineamento: Foram analisados, retrospectivamente, prontuários de pacientes com achados microbiológicos positivos para infecções por actinomicetos. Local do estudo: Um hospital universitário de atendimento terciário em Porto Alegre, Rio Grande do Sul, Brasil. Pacientes e métodos: Foram incluídos neste estudo, pacientes com diagnóstico de actinomicose, nocardiose e rodococose, num período de 1978 a 2009. Os critérios microscópicos para o diagnóstico de actinomicetose foram os seguintes: actinomicose - composto por grânulos actinomicóticos, filamentos Gram-positivos, não ácido-resistentes; nocardiose - bactérias filamentosas ramificadas, Gram-positivas, e ácido-resistentes; rodococose - cocobacilos Gram-positivos, ácido resistentes. Resultados: Foram incluídos 59 pacientes com actinomicetose. Actinomicose foi obervada em 27 pacientes entre 8 e 65 anos (idade média de 39,9 anos), 22 do sexo masculino (81,5%). Doença oral (cárie dentária, a doença periodontal) esteve frequentemente associada, sendo procedimento odontológico o fator de risco mais importante. A apresentação clínica foi actinomicose torácica em 24 casos, em dois facial e em um cérvico-facial e mediastinal. O diagnóstico microscópico foi positivo em 25, com o isolamento do organismo em cultivo anaeróbico em um, e, pelo teste de imunofluorescência direta em um. Estes dois últimos casos foi identificado como A. israelii. O tratamento mais utilizado consiste na administração prolongada de penicilina e esteve associado a boa evolução na maioria dos casos. Nocardiose foi observada em 27 pacientes, a idade variou entre 21 e 84 anos, idade média de 51,8 anos. A manifestação mais comum foi pneumonia cavitária, apresentado no paciente imunossuprimido, especialmente recebendo altas doses de corticoterapia. Todos os casos foram positivos para filamentos bacterianos ramificados Gram-positivos, ácido resistentes, sugestivos de espécies de Nocardia. Nocardia sp foi isolada em 14 casos, ―N. asteroides” em 7, N. farcinica em 2, N. brasiliensis em 1, N. pseudobrasiliensis em 1, N. abscessus em 1 e N. cyriacigeorgica em 1. Doze pacientes foram a óbito e os restantes tiveram melhora clínica. A rodococose foi diagnosticada em 5 pacientes, com idade, no momento do diagnóstico, de 22-69 anos (média de 45,6). Rhodococcus foi isolado em todos os 5 casos, três pacientes imunodeprimidos apresentaram infecção pulmonar pelo R. equi. O caso do paciente com HIV/AIDS foi fatal. Conclusões: Esta experiência, indica que a informação clínica associada ao Gram e a ácido-resistência em amostras clínicas é útil no reconhecimento da infecção por actinomicetos. A actinomicetose deve ser sempre considerada em pacientes apresentando doença febril supurativa ou radiografia de tórax anormal, em paciente com estado imune alterado causado por determinadas drogas (corticoterapia) e condições associadas (HIV/AIDS).Background: Diseases caused by pathogenic aerobic and facultative anaerobic actinomycetes differ considerably with respect to their etiology, pathogenesis, clinical appearence and epidemiology. Objectives: To analyse the age distribution, clinical manifestations, underlying diseases and associated medical conditions, radiographic findings, microbiology, treatment and outcome, in patients with actinomycetosis (actinomycosis, nocardiosis, rhodococcosis). Design: The medical records of patients with positive microbiology findings to actinomycetes infections were retrospectively analysed. Settings: A university-based tertiary care hospital in Porto Alegre, Rio Grande do Sul, Brazil. Patients and methods: From 1978 through 2009 patients diagnosed with actinomycosis, nocardiosis, and rhodococcosis were included in this study. The microscopic criteria for diagnosis of actinomycetosis were as follow: actinomycosis –granules composed by branching Gram-positive organisms non acid-fast stained; nocardiosis - branched filamentous, Gram-positive, and acid-fast bacteria; rhodococcosis - coccobacilli Gram-positive, and acid-fast organism. Results: Sixty-five patients with actinomycetosis were included. Actinomycosis was oberved in 27 patients between 8 and 65 years old (mean age, 39,9 years), 22 were male (81,5%). Oral disease (poor dentition, periodontal disease) frequently associated with dental procedure was the most important risk factor. The clinical presentation was thoracic actinomycosis in 24 cases, facial in two, and cervico-facial and mediastinal one. Microcopic diagnosis were positive in 25, recovery of organism in anaerobic culture in one, and by fluorescent antibody test in one. These last two cases was identified as A. israelii. Treatment most commonly consisted of prolonged administration of penicillin and was associated with good outcome in the majority of cases. Nocardiosis was observed in 27 patients, aged 21 to 84 years old, with a mean age of 51,8 years. Cavitary pneumonia was the most common manifestation, presented in immunosuppresed patient, especially receiving high-dose corticotherapy. All cases were positive for branching Gram-positive, acid-fast bacterial filaments, suggestive of a Nocardia species. Nocardia sp was isolated in 14 cases, ―N. asteroides” in 7, N. farcinica in 2, N. brasiliensis in 1, N. pseudobrasiliensis in 1, N. abscessus in 1 and N. cyriacigeorgica in 1. Twelve patients died and the remaining cases were well improved. The diagnosis of rhodococcosis was made in five patients, ranged in age at time of diagnosis from 22 to 69 years, with a mean age of 45,6 years. Rhodococcus was isolated in all 5 cases, three immunocompromised patients showed pulmonary infection by R. equi. The case with HIV/AIDS was fatal. Conclusions: This experience, indicates that clinical information with Gram and acid-fast stains on clinical specimens is helpful in recognizing the possibility os actinomycetes should always be considered as a cause os suppurative febrile illness or abnormal chest roentgenograms in patient who may have an altered immune status caused by certain drugs (corticotherapy) and underlying conditions (HIV/AIDS).
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Rothhaar, Eric. « Zur Entwicklung der Empfindlichkeit von Rhodococcus equi gegenüber Antibiotika ». [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=980904633.
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Cornelis, Karen. « Behaviour of the phytopathogenic bacterium Rhodococcus fascians on plants ». Doctoral thesis, Universite Libre de Bruxelles, 2000. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/211753.
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Kutanovas, Simonas. « Investigation of tetramethylpyrazine degradation in Rhodococcus sp. TMP1 bacteria ». Doctoral thesis, Lithuanian Academic Libraries Network (LABT), 2013. http://vddb.laba.lt/obj/LT-eLABa-0001:E.02~2013~D_20130701_092345-19015.
Texte intégralRésumé :
The catabolism of alkylpyrazines is poorly described. The pathways for the degradation of di- and tri-substituted pyrazines have been proposed, but these related routes consistently include a hydroxylation step that cannot be performed on tetramethylpyrazine. Here we describe for the first time the catabolic pathway of tetramethylpyrazine in tetramethylpyrazine-degrading Rhodococcus jostii TMP1 strain. MS/MS analysis of the protein primarily upregulated by tetramethylpyrazine led to the identification of the gene locus encoding proteins required for the initial steps of tetrametylpyrazine degradation and for the regulation of this locus. Tetramethylpyrazine degradation starts with oxidative ring cleavage catalysed by monooxygenase TpdAB, which produces (Z)-N,N'-(but-2-ene-2,3-diyl)diacetamide. This compound is further hydrolysed by amidase TpdC to N-(3-oxobutan-2-yl)acetamide. TpdE was confirmed to be an aminoalcohol dehydrogenase yielding N-(3-hydroxybutan-2-yl)acetamide. By determining intermediates, enzymes involved and genes responsible for tetramethylpyrazine degradation we provide the first validated pathway for pyrazine degradation. We also report that Rhodococcus jostii TMP1 is capable of modifying various alkylpyrazines and alkylpyridines and can be employed for the bioconversion of 2,4,6-trimethylpyridine and 2,4,6-trimethylpyridin-3-ol biosynthesis.Alkilpirazinų katabolizmas bakterijose yra prastai ištirtas. Nors yra žinomi tarpiniai metabolitai susidarantys skaidant di- ir tri- pakeistus alkilpirazinus, tačiau šių junginių skaidymas prasideda hidroksilinimo reakcija, kuri negalima tetrametilpirazino atveju. Šiame darbe pirmą kartą nustatytas tetrametilpirazino katabolizmo kelias šį junginį skaidančiose Rhodococcus jostii TMP1 bakterijose. MS/MS de novo sekoskaitos būdu identifikavus tetrametilpirazinu indukuojamą baltymą, buvo nustatyta genų sankaupa, koduojanti pradines tetrametilpirazino katabolizmo reakcijas katalizuojančius fermentus ir transkripcijos reguliatorių, dalyvaujantį šių genų aktyvavime. Pradiniame tetrametilpirazino skaidymo etape monooksigenazė TpdAB katalizuoja oksidacinį žiedo atidarymą, susidarant (Z)-N,N'-(but-2-ene-2,3-diil)diacetamidui. Tolesnę skaidymo reakciją katalizuoja amidazė TpdC, kurios produktą N-(3-oksobutan-2-il)acetamidą aminoalkoholių dehidrogenazė TpdE redukuoja iki N-(3-hidroksibutan-2-il)acetamido. Nustačius tarpinius tetrametilpirazino skaidymo metabolitus, reakcijas katalizuojančius fermentus ir juos koduojančius genus buvo rekonstruotas pirmasis alkilpirazinų katabolizmo kelias bakterijose. Darbo metu taip pat parodyta, kad Rhodococcus jostii TMP1 bakterijos modifikuoja daugelį alkilpirazino ir alkilpiridino junginių ir gali būti panaudotos 2,4,6-trimetilpiridin-3-olio biosintezei iš 2,4,6-trimetilpiridino.
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Harris, Randall. « Degradation of dichloroalkanes by Rhodococcus rhodochrous and Pseudomonas oleovorans ». Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=98965.
Texte intégralRésumé :
The degradation of C8-C16 alpha,beta-dichloroalkanes by Rhodococcus rhodochrous ATCC 13808 and Pseudomonas oleovorans ATCC 29347 was studied. The carbon chain length of the alpha,beta-dichloroalkane influenced the degradation rates of these compounds and the observed trends were species-dependent. R. rhodochrous exhibited faster rates towards the longer-chained compounds whereas P. oleovorans most rapidly degraded the shorter-chained compounds. This observation is consistent with the chain-length specificity of the hydroxylase enzymes responsible for initiating the degradation of alkanes by each organism. Studies conducted in a cyclone batch reactor indicated that alpha,beta-dichloroalkanes are modified to chlorinated metabolites prior to the dechlorination step. This indicates that the degradation of alpha,beta-dichloroalkanes is initiated by a hydroxylase acting on the non-chlorinated end of the molecule.
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Sun, Lingshuang. « RHODOCOCCUS EQUI INFECTION AND INTERFERON-GAMMA REGULATION IN FOALS ». UKnowledge, 2012. http://uknowledge.uky.edu/gluck_etds/7.
Texte intégralRésumé :
Rhodococcus equi (R. equi) is one of the most serious causes of pneumonia in young foals. The clinical disease is of great concern to breeding farms worldwide due to the impact of mortality on economic losses. While adult horses are resistant to R. equi, foals exhibit a distinct age-associated susceptibility. The mechanism underlying this susceptibility in foals is not well understood. Interferon-gamma (IFNg) plays an important role in the clearance of R. equi, but its expression is impaired in neonatal foals. Moreover, the regulation of this age-related IFNg expression in foals remains unknown. In humans, IFNg expression has been shown to be regulated by DNA methylation, lymphoproliferation, and influenced by environmental exposure. Therefore, we hypothesized that environmental exposure promotes IFNg expression through regulation of DNA methylation and lymphoproliferation. The objectives were: (1) to estimate the relevance of IFN-g production and R. equi infection in foals; (2) investigate the role of lymphoproliferation and DNA methylation in the regulation of IFN-g expression in foals; (3) to evaluate the effect of environmental exposure on IFN-g expression by housing foals in a barn environment verses pasture.; (4) to investigate the effect of environment exposure on antigen-presenting cells (APC), which sensor the environmental antigens and modulate IFN-g production by T cells. The results demonstrated that the IFN-g expression was inversely correlated with the age-related susceptibility to R. equi infection. lymphoproliferation promoted IFN-g expression in foals, whereas, DNA methylation repressed IFN-g expression. The IFN-g expression was augmented in foals exposed to the barn air which contained higher numbers of aerosol miroorganisms. DNA on the IFN-g promoter was demethylated and the lymphoproliferative activity was elevated in foals with barn-air exposure. The barn-air exposure also promoted the maturation and activation of APC to prime IFN-g expression by T cells in foals. Overall, this body of work demenstrated a relationship between IFN-g expression and R. equi infection, provided novel information on mechanisms that regulate IFN-g expression, and identified the effect of environment on mechanisms responsible for IFN-g expression.
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Chen, S. « Organisation and regulation of naphthalene catabolic genes in Rhodococci ». Thesis, Queen's University Belfast, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.403265.
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Snell, David Alfred. « The application of Rhodococcus sp. AJ270 as a biocatalyst ». Thesis, University of Sunderland, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285282.
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Kariptas, Ergin. « Chemical composition of Rhodococcus ruber with different growth conditions ». Thesis, University of Newcastle Upon Tyne, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.340719.
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Fosdike, William L. J. « The alkene monooxygenase from the Rhodococcus rhodocurous B-276 ». Thesis, University of Warwick, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.247446.
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Hussain, Sajad. « Enantioselective hydrolysis of phenylglycineamide to phenylglycine by Rhodococcus NP3854 ». Thesis, Keele University, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.288510.
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Cahill, Matthew Jameson. « Sequencing and assembly of the Rhodococcus aetherivorans I24 genome ». Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608824.
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Far, Pierre D. « Characterisation of a nonribosomal peptide synthetase from Rhodococcus I24 ». Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613722.
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Anastasi, Elisa. « Comparative genomics and emerging antibiotic resistance in Rhodococcus equi ». Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25887.
Texte intégralRésumé :
Rhodococcus equi is a soil-dwelling facultative intracellular pathogen that can infect many mammals, including humans. R. equi is most well known for its ability to cause severe pyogranulomatous disease in foals, primarily involving the lungs although other body systems may also be affected. The disease is endemic on many horse-breeding farms worldwide and poses a severe threat to the horse breeding industry because there is no vaccine available. Current prophylaxis is based on systematic preventative treatments with macrolides combined with rifampicin, which are also used to treat clinical cases of the disease in foals. In this thesis I have used a combination of wet laboratory and bioinformatic approaches to identify the molecular basis of emerging combined resistance to macrolides and rifampicin in R. equi foal isolates from the USA. The genomes of a selection of resistant and susceptible strains from across the USA were sequenced and assembled. Resistance genes were systematically searched by reciprocal best-match BLASTP comparisons to known antibiotic resistance determinants. This led to the discovery of a novel erythromycin ribosomal methylase (erm) gene, erm(46), in all resistant strains. Complementation analysis in a susceptible R. equi strain showed that erm(46) was sufficient to confer resistance to all macrolides, lincosamides, and streptogramin B. The erm(46) gene is carried by an integrative conjugative element (ICE) which is transferable between R. equi strains. The ICE is formed by two distinct parts, a class I integron associated with an IS6100 sequence and the erm(46) determinant carried by a sub-element which contains putative actinobacterial conjugative translocase apparatus and a transposase/integrase. All resistant strains also carry the same non synonymous point mutation in rpoB conferring rifampicin resistance. Thus, these strains are carrying double resistance to the most commonly used antibiotics to treat R. equi worldwide. Phylogenetic analysis based on the core genome demonstrated that all resistant strains are clonal. This indicates that although conjugal acquisition of the erm(46) conjugative element may occur at a high frequency, the need for the concurrent presence of a second rpoB mutation for survival in the macrolide and rifampicin dominated farm environment has effectively selected for the spread of a single clone. In the second section of this work, we sequenced a further 20 R. equi genomes from difference sources (equine, porcine, bovine, human), including representatives of each of the seven major genogroups previously defined in our laboratory based on pulsed field gel electrophoresis. I have used the newly acquired genetic information to study the genome of R. equi and analyse its diversity within and outwith its species group. This enabled us to explore the pan genome and define that R. equi is a genetically well-defined bacterial species. Our results provide definitive evidence that resolves the current dispute over R. equi classification, specifically they do not support the recent proposal (based on classical polyphasic bacterial taxonomical methods) that R. equi should be transferred to a new genus. Our core-genome phylogenomic analyses unambiguously show that the genus Rhodocococcus is monophyletic and that R. equi forms a clade together with the most recently described related environmental species R. defluvii that radiates from within the genus. Together with other shared biological and genetic characteristics, namely the unique niche-adaptive mechanism based on evolutionarily related extrachromosomal replicons, R. equi should be conseidered a bona fide member of the genus Rhodococcus. We also confirm that Rhodococcus spp. and Nocardia spp. are sufficiently distinct to warrant them belonging to different genera. In conclusion, this work used whole genome sequencing to characterize the molecular basis underlying the emergence and clonal spread of multi-resistant R. equi in horse breeding farms in the USA. This work also highlights the limitations of classical taxonomical approaches in bacterial systematics, and illustrates the importance of incorporating modern phylogenomic approaches to understand the evolutionary relationships between bacterial strains and their accurate taxonomic position.
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Azman, Hazeeq. « Bioligninolysis : degradation of ionic liquid derived lignin by Rhodococcus ». Thesis, Imperial College London, 2015. http://hdl.handle.net/10044/1/44278.
Texte intégralRésumé :
There has been much recent interest in using ionic liquids for processing lignocellulosic biomass. While cellulose has an acknowledged application in generating biofuels, it would be valuable to use the abundant lignin present as well. Rhodococcus has been reported previously to degrade lignin. Therefore, it is attractive to consider a scheme in which an ionic liquid is also used to enhance the microbial breakdown of lignin (bioligninolysis). By using vanillic acid as model compound (Chapter 3), results showed that Rhodococcus UKMP-5M is able to degrade vanillic acid as a sole carbon source at 10mM concentration to give the highest growth rate. An oxygen-dependent reaction degrades vanillic acid into protocatechuic acid and formate in a previously undescribed metabolic pathway. In Chapter 4, GC-MS demonstrated guaiacol as the major product of lignin degradation and the lignin degradation assay indicates that the treatment of lignin with ionic liquids assist the lignin degradation despite some ionic liquids showing a toxicity effect on the cells. Toxicological studies (Chapter 5) demonstrated different ionic liquids show varying toxicity to the bacteria. By using classical disk diffusion test in screening 16 different ionic liquids, it was revealed that the toxicity is correlated with the size of the ionic alkyl chain; however, the carbon atom count, not the structure or the distribution of those atoms in the cation, correlates directly with the toxicity. The strongest link was discovered with pH effects rather than with structure in the toxicity of acidic ionic liquids. We also propose that the octanol-water partition coefficient (Kow) has the controlling impact on the toxicity of ionic liquids. Bacterial growth curves exhibited three different trends: a complete inhibition, an increase in toxicity with an increase of ionic liquid concentration and the extension of the lag phase due to bacterial adaptation. This study established that Rhodococcus UKMP-5M could adapt and grow in the presence of ionic liquids with 1-ethyl-3-methylimidazolium acetate, [Emim][OAc] as the most promising candidate in designing an ionic liquid-facilitated system of bioligninolysis.