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.

Environmental samples (air and soil) were collected from thoroughbred breeding farms with different prevalences of R. equi pneumonia to increase our understanding of the ecology of virulent R. equi on horse farms. The airborne population of virulent R. equi was a major focus of this research, as inhalation of the pathogen from the environment is considered the primary route of pulmonary infection. Air sampling was performed using an air monitoring system with selective media to facilitate the recovery of R. equi, allowing quantitative measurement of airborne virulent R. equi. Polymerase chain reaction and DNA hybridisation techniques were used to evaluate environmental samples to identify and differentiate R. equi.

Tierärztl. Hochsch., Diss., 2004--Hannover.

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-12-02Bitstream added on 2014-06-13T18:44:31Z : No. of bitstreams: 1 silva_p_dr_arafcf.pdf: 2320520 bytes, checksum: 5b67cf713d666bdba286bd22e1e7c114 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As bactérias Gram-positivas que contêm ácidos micólicos na parede celular estão classificadas no grupo dos actinomicetos aeróbios ou bactérias corineformes e nocardioformes. Nesse grupo encontra-se o Rhodococcus equi, o qual é relevante à medicina veterinária e humana tal como as micobactérias, causando doença pulmonar que pode mimetizar casos de tuberculose. R. equi é considerado como agente patogênico em potros e tem emergido como oportunista em humanos, especialmente, associado à infecção pelo vírus da imunodeficiência humana. Assim como nos animais, a rodococose humana afeta principalmente, os pulmões, com características clínicas e patológicas, similares à tuberculose pulmonar, em pacientes imunocomprometidos ou não. A identificação de Rhodococcus equi pode ser realizada com base numa variedade de características, fenotípicas, genotípicas e técnicas cromatográficas. Morfologia das colônias, morfologia celular e resistência parcial ao álcool ácido são características chaves para a caracterização inicial. R. equi não oxida ou fermenta carboidratos e nem utiliza acetato, citrato e malonato, como única fonte de carbono, produz catalase, o fator equi (teste de CAMP) e lipase. Não produz amilase, b-galactosidase (ONPG), casease, DNase, esculinase, gelatinase, H2S, indol, lecitinase e oxidase. Demonstra comportamento variável para as provas de nitrato redutase, urease e redução do hipurato, decompõe a adenina, mas não hipoxantina, tirosina e xantina. O método molecular para a identificação de R. equi utiliza a PCR para amplificar um fragmento de 959 pares de base do gene choE, o qual codifica a enzima cholesterol oxidase (COX). Na identificação química, semelhante às espécies do gênero Mycobacterium, membros do gênero Rhodococcus contêm ácidos micólicos plausíveis de serem identificados pela cromatografia... / Gram-positive bacteria, containing mycolic acids in the cellular wall are classified in aerobic actinomycetes or corineform and nocardioform bacteria group. Rhodococcus equi is included in this group, and it is very important to the veterinary and human medicine. Rhodococcus equi is a well-recognized bacterial pathogen in veterinary medicine. First isolated from foals, it causes an important chronic granulomatous pneumonia and lung abscesses. The infection also occurs in humans, often following immunosuppression of various causes. The increased number of human cases reported recently is partly the result of the spread of AIDS but may also reflect the increasing awareness by medical laboratories of this opportunistic pathogen and their improved ability to identify it rather than to dismiss it as a contaminating micrococcus or diphtheroid. R. equi can be identified on the basis of a variety of conventional phenotypic characteristics including microscopic (Gram and acidfast staining) and macroscopic morphologies, growth requirements, metabolism of glucose, and phenotypic molecular characteristics including the presence of mycolic acid composition, which is detected by thin-layer chromatography. The colonial morphology of R. equi is diverse and consists of three major varieties: pale pink and slimy, coral and non-slimy, and pale yellow in color, non-slimy. Colorless colonial variants may also occur. R. equi is a non-motile gram-positive pleomorphic coccobacillus, varying from distinctly coccoid to bacillary depending on growth conditions. All of the rhodococci from clinical specimens are generally weakly acid fast when stained either by the modified Kinyoun method or by the Ziehl-Neelsen method. Regarding biochemical characteristics, the organism is generally biochemically unreactive. It fails to oxidize or ferment carbohydrates, neither uses sodium... (Complete abstract click electronic access below)

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.

Die Isolierung von Phagosomen ermöglicht die biochemische Analyse der Phagosomen-Zusammensetzung sowie der an der Phagosomenreifung beteiligten Moleküle. Deshalb wurde im Rahmen dieser Promotionsarbeit eine Methode entwickelt, die es ermöglicht, Bakterien-enthaltende Phagosomen zu isolieren. Diese Methode erzielt im Vergleich zu anderen in der Literatur beschriebenen Methoden eine gute Ausbeute (fast 40 Prozent) und vor allem eine höhere Reinheit an Bakterien-enthaltenden Phagosomen. So besteht keine Kontamination mit Teilen des Golgi-Apparates und nur eine sehr geringe Kontamination mit endosomalen und lysosomalen Proteinen sowie Plasmamembranbestandteilen. Allerdings wurde eine Kontamination mit Mitochondrien und ER detektiert. Letzteres muss nicht unbedingt eine Kontamination darstellen, sondern könnte ein wichtiger Bestandteil von Phagosomen sein. Afipia felis ist ein Gram-negatives Bakterium, das für einige Fälle der Katzen-Kratz Krankheit verantwortlich ist. Es kann innerhalb von Makrophagen überleben und sich vermehren. Die genaue Kompartimentierung der Afipia felis-enthaltenden Phagosomen in Makrophagen war allerdings unbekannt und sollte deshalb in der vorliegenden Promotionsarbeit analysiert werden. Ovalbumin Texas Red, mit dem Lysosomen vor der Infektion markiert wurden, gelangt nicht in die Afipien-enthaltenden Phagosomen, und die Afipien-enthaltenden Phagosomen sind auch nicht zugänglich für Ovalbumin Texas Red, mit dem das gesamte endozytische System nach der etablierten Infektion markiert wurde. Außerdem sind etablierte, isolierte Afipia felis-enthaltende Phagosomen nur in geringem Umfang positiv für spät endosomale/lysosomale Markerproteine und negativ für früh endosomale Markerproteine. Die Afipien, die ein nicht endozytisches Kompartiment etablieren, werden vom Makrophagen in ein EEA1-negatives Kompartiment aufgenommen, das auch zu späteren Zeitpunkten negativ für LAMP-1 ist. Nur die circa 30 Prozent der Afipien, die sich in einem Kompartiment befinden, das zum endozytischen System gehört, gelangen nach der Aufnahme durch den Makrophagen in ein EEA1-positives Kompartiment, das zu einem späteren Zeitpunkt positiv für LAMP-1 wird. Tötung der Afipien oder Opsonisierung mit Antikörpern vor der Infektion normalisiert die Reifung der Afipia felis-enthaltenden Phagosomen in den J774E-Makrophagen. Somit konnte nachgewiesen werden, dass die Mehrzahl der Phagosomen (70 Prozent), die Afipia felis enthalten, nicht zum endozytischen System gehören. Diese ungewöhnliche Kompartimentierung besteht bereits bei der Aufnahme und kann nur von lebenden Afipien etabliert werden. Rhodococcus equi ist ein Gram-positives Bakterium, das unter anderem Bronchopneumonien beim Fohlen verursacht. Aber auch Menschen und andere Säugetiere sind von Infektionen mit R. equi betroffen. Die Fähigkeit der Rhodokokken, innerhalb der Makrophagen zu überleben und sich zu vermehren, ist mit dem Vorhandensein eines 85 kbp Plasmids assoziiert. Da über die genaue Kompartimentierung von R. equi im Mausmakrophagen wenig bekannt war, und der Frage, ob es einen Unterschied zwischen der Kompartimentierung von R. equi(+)- und R. equi(-)-enthaltenden Phagosomen gibt, noch nicht nachgegangen wurde, war beides Thema dieser Promotionsarbeit. Dabei zeigt sich, dass R. equi(-)-enthaltende Phagosomen wesentlich stärker mit den spät endosomalen/lysosomalen Markerproteinen vATPase und LAMP-1 assoziiert sind sowie eine höhere ß-Galaktosidase-Aktivität aufweisen als die R. equi(+)-enthaltenden Phagosomen. Da sowohl die isolierten R. equi(-)- als auch die R. equi(+)-enthaltenden Phagosomen mit dem früh endosomalen Markerprotein rab5 assoziiert sind, ist anzunehmen, dass Rhodokokken unabhängig vom Vorhandensein des 85 kbp Plasmids in der Lage sind, die Phagosomenreifung zu verzögern. Aber R. equi(-) kann die Reifung zwar verzögern, aber letztendlich nicht verhindern. Wahrscheinlich reifen die Phagosomen, die R. equi(-) enthalten, zu einem späteren Zeitpunkt zu Phagolysosomen, wohingegen R. equi(+) ein ungewöhnliches Kompartiment etabliert und dadurch die Phagosomenreifung endgültig zu verhindern scheint. Somit ist anzunehmen, dass mindestens ein vom 85 kbp Plasmid kodiertes Molekül für die Etablierung dieses ungewöhnlichen, R. equi(+)-enthaltenden Kompartimentes, verantwortlich ist. Da eine Infektion mit Rhodococcus equi zytotoxisch für die infizierte Zelle sein kann, wurde die von den Rhodokokken vermittelte Zytotoxizität näher analysiert. Die in dieser vorliegenden Promotionsarbeit dargestellten Ergebnisse zeigen deutlich, dass nur die Plasmid-enthaltenden Rhodokokken zur Nekrose, aber nicht zur Apoptose ihrer Wirtszellen führen, während R. equi(-) keinen Einfluss auf die Vitalität ihrer Wirtszellen haben. Dieses Phänomen ist allerdings abhängig vom Wirtszelltyp. So sind R. equi(-) als auch R. equi(+) für humane Monozyten nur geringfügig zytotoxisch. / The isolation of phagosomes from phagocytes enables their fine biochemical analysis as well as the determination of the role of various molecules in phagosome biogenesis. Therefore, a method to purify bacteria-containing phagosomes from infected macrophages has been established in this study. In comparison with previously described methods a good yield (40 per cent) and a higher level of purity of bacteria-containing phagosomes were obtained using this phagosome isolation method. No Golgi-derived contamination and only very little endosomal or lysosomal and plasma membrane-derived contaminations was found. Furthermore, some mitochondrial and ER contaminations were detectable. Afipia felis is a Gram-negative bacterium that causes some cases of human Cat Scratch Disease. It can survive and multiply in macrophages, but the precise intracellular compartimentalization of Afipia felis-containing phagosomes is unknown. In this study we show evidence, that 70 per cent of Afpia felis-containing phagosomes do not belong to the endocytic pathway. This is supported by the facts that neither did ovalbumin preloaded into lysosomes enter most Afipia felis-containing phagosomes, nor did ovalbumin loaded into the endocytic system after infection. Furthermore the percentage of isolated Afipia felis-containing phagosomes positive for late endosomal/lysosomal markerproteins were very low and early endosomal marker proteins were rarely detectable. Those bacteria that were to be found in a nonendosomal compartment entered the macrophage via an EEA1-negative compartment, which remained negative for LAMP-1. The small population of Afipia felis whose phagosomes were part of the endocytic system entered into an EEA1-positive compartment which also subsequently acquired LAMP-1. Killing of Afipia felis or opsonization with antibodies before infection lead to a strong increase in the percentage of Afipia felis-containing phagosomes that interact with the endocytic system. We conclude that most phagosomes containing Afipia felis are disconnected from the endocytic system. This unusual compartalization is decided at uptake and can only be established by viable Afipia felis. Rhodococcus equi is a Gram-positive bacterium that causes granulomatous pneumonia in foals. It is also a pathogen for other animals and human beings. The pathogenicity of Rhodococcus equi is depending on its ability to exist and multiply inside macrophages and this correlates with the presence of a 85 kbp plasmid. The aim of this study was to elucidate the intracellular compartmentation of Rhodococcus equi and the mechanism by which the bacteria might avoid the destruction in host macrophages. The importance of the virulence-associated plasmid was also evaluated. In this study it is shown that R. equi(-)-containing phagosomes contained much more of the late endosomal/lysosomal marker proteins vATPase or LAMP-1 and also a larger amount of the lysosomal enzyme ß-galactosidase than R. equi(+)-containing phagosomes. Both R. equi(+)- and R. equi(-)-containing phagosomes associated with the early endosomal marker protein rab5. Based on these results it can be speculated that Rhodococcus equi is able to slow down or arrest the maturation of its phagosome independently of the 85 kbp virulence-associated plasmid. Whereas R. equi(-) is able to slow down but not to arrest its phagosomes, R. equi(+) establishes an unusual compartment, which possibly represents a recycling-endosome. Therefore, for the long term establishment of the unusual R. equi(+)-containing phagosomes at least one of the molecules encoded by the 85 kbp plasmid is necessary. Since Rhodococcus equi infection ultimately proves toxic for macrophages, the R. equi mediated cytotoxicity was analyzed. In this study it is shown that Rhodococcus equi induce necrosis in their host cells and which is dependent on the presence of the virulence-associated 85 kbp plasmid. But the Rhodococcus induced necrosis can only be observed in mouse macrophages and not in human monocytes.

Rhodococcus equi is a Gram-positive intracellular pathogen which can cause severe bronchopneumonia in foals. In recent years, the role of this bacterium as human pathogen has been noted, as R.equi infections in humans have increase in frequency. This increase is associated with the rise in immunosupressed individuals, specially AIDS patients, where infection leads to symptoms and pathology similar to those seen in foals with a high mortality rate. Due to its capability to survive and multiply in murine and equine macrophages, R.equi has been classified as a facultative intracellular bacterium. R.equi is found frequently in macrophages in alveolar infiltrate from infected animals. The pathogenicity of R.equi depends on its ability to exist and multiply inside macrophages and has been associated with the presence of virulence plasmids. It has been observed that, inside foal alveolar macrophages, R.equi-containing vacuoles (RCVs) do not mature into phagolysosomes. However, most of the intracellular events during R.equi infection have not been investigated in detail. The aim of this study was to elucidate the intracellular compartmentation of R.equi and the mechanism by which the bacteria avoid destruction in host macrophages. The importance of the virulence-associated plasmids of R.equi for the establishment of RCVs was also evaluated. Furthermore, the intracellular fate of viable and non-viable R.equi was compared in order to study whether viability of R.equi influeciantes the establishment of RCVs. In this study, the RCV was characterized by using a variety of endocytic markers to follow the path of the bacteria trhough murine macropages. Transmission electron microscopy-base analysis showed that R.equi was found equally frequently in phagosomes with loosely or thightly apposed membranes, and RCV often contains numerous membranous vesicles. Laser scanning microscopy of infected macrophages showed that the majority of phagosomes containing R.equi acquired transiently the early endosomal markers Rab5, Ptlns3P, and EEA-1, suggesting initially undisturbed phagosome maturation. Although the RCV acquired some late endosomal markers, such as Rab7, LAMP-1, and Lamp-2, they did not acquired vATPase, did not interact with pre-labeled lysosomes, and failed to acidify. These data clearly suggest that the RCV is a compartment which has left vacuoles that resemble multivesicular body compartments (MVB), which are transport intermediates between early and late endosomes and display internal vesicles very similar to the ones observed within RCVs. Analyisis of several R.equi strains containing either VapA- or VapB-expressing plasmids or neither demonstrated that the possession of the virulence-associated plasmids does not affect phagosome trafficking over a two hour period of infection. The finding that non-viable R.equi was still able to inhibit phagosome maturation (although not to the same extent as viable R.equi did) suggests that heat-insensitive factors, such as cell periphery lipids, may play a major role in inhibition of phagosome maturation, although heat-sensitive factors may also be involved. / Rhodococcus equi ist ein Gram-positives, fakultativ intrazelulläres Bakterium, das unter anderem die Ursache von Bronchopneumonien bei Fohlen ist. Menschen und andere Säugetiere können ebenfalls von Infektionen mit R. equi betroffen sein. In den letzten Jahren ist die Häufigkeit klinischer Infektionen mit R. equi bei Menschen gestiegen. Die wachsende Anzahl an mmunosupprimierten Patienten (hauptsächlich AIDS-Patienten) liegt dieser Zunahme an Infektionen zugrunde. Die Symptomatologie und Pathologie der Infektion mit R. equi ist bei AIDS-Patienten und Fohlen ähnlich. Die Sterblichkeitsrate ist in beiden Fällen hoch. Die Fähigkeit der Rhodokokken, innerhalb von Makrophagen zu überleben und sich zu vermehren, ist mit dem Vorhandensein von Virulenzplasmiden (virulence-associated plasmids) verbunden. Innerhalb des Makrophagen befinden sich die Rhodokokken in einem Phagosom, das nicht mit Lysosomen fusioniert. Die genaue Kompartimentierung der Rhodococcus equi-enthaltenden Phagosomen in Makrophagen war bisher unbekannt und wurde deshalb in der vorliegenden Promotionsarbeit untersucht. Mit Hilfe mehrerer endozytischer Marker wurde das R. equi-enthaltende Kompartiment charakterisiert. Mögliche Unterschiede zwischen der Kompartimentierung von R. equi(+)- und R. equi(-)-enthaltenden Phagosomen ist ebenfalls Thema dieser Promotionsarbeit. Weiterhin wurde die Etablierung des phagosomalen Kompartiments für jeweils lebende und tote Rhodokokken verglichen. Transmissionselektronenmikroskopische Analysen haben gezeigt, dass die Phagosomenmembran Rhodococcus equi-enthaltender Phagosomen sowohl locker als auch eng anliegend sein kann (50%). Darüber hinaus wurden häufig zahlreiche, membranöse Vesikel in R. equi-enthaltenden Phagosomen gefunden. Diese Phagosomen zeigen somit Ähnlichkeiten zu Multivesicular Bodies. Multivesicular Bodies sind intermediäre Kompartimente zwischen frühen und späten Endosomen und zeigen ebenfalls eine Vielzahl von internen Vesikeln. Untersuchungen am konfokalen Lasermikroskop ergaben, dass die Mehrheit der R. equi-enthaltenden Phagosomen die früh endosomalen Marker Rab5, PtIns3P und EEA-1 transient akquirieren. Dieser Befund deutet auf eine ungestörte phagosomale Reifung im frühen Stadium hin. Trotz der beobachteten Akquisition der spät endosomalen Marker Rab7, LAMP-1 und LAMP-2 konnte keine Akquisition der vATPase, keine Interaktion mit vormarkierten Lysosomen und keine Ansäuerung von R.equi-enthaltenden Phagosomen nachgewiesen werden. Diese Ergebnisse weisen darauf hin, dass R. equi-enthaltende Phagosomen das früh endosomale Stadium abschließen, aber einen typisch spät endosomale Zustand nicht erreichen. Die Analyse unterschiedlicher R. equi-Stämme, die entweder vapA- oder vapB-exprimierende Virulenzplasmide enthalten, hat gezeigt, dass die Anwesenheit von Virulenzplasmiden die phagosomale Reifung über eine Infektionsperiode von zwei Stunden nicht beeinflusst. Getötete Rhodokokken waren in der Lage, die phagosomale Reifung zu inhibieren, aber in geringerem Ausmaß als lebende Rhodokokken. Das weist darauf hin, dass hitze-insensitive Faktoren (wie zum Beispiel Lipide der Zellwand) zur Inhibierung der phagosomalen Reifung entscheidend sind, obwohl dazu auch hitze-sensitive Faktoren (wie Proteine) relevant sein können.

Bioremediation tends to be an effective technology, used to remove pollutants and it involves relatively little capital. Microorganisms, especially bacteria, evolving into many types with exceedingly diverse metabolic capabilities, which can be applied in bioremediation. The two bacterial strains, Nocardioides simplex FJ2-1A and Rhodococcus opacus HL PM-1 are capable of biodegradation of 2,4,6-trinitrophenol (TNP, picric acid). In N. simplex, the biodegradation pathway is inducible while in R. opacus is constitutive. Some enzymes were identified in both strains and shown to be isofunctional. In this work, a molecular characterisation of the orfB from the npd gene cluster was achieved. The mutant R. opacus AR1 strain deficient in the orfB gene was constructed by a gene replacement method using the expression of sacB as the selectable marker. The gene was also cloned as the His tag fusion protein and expressed in E. coli. Theoretical pI / Mw predictions for the protein sequence were 5.47 / 42536.23 Da. SDS-PAGE analysis showed the size of the purified fusion protein, which was calculated to be 42.5 kDa. The protein was purified by Ni-NTA metal affinity chromatography. This study furthermore identified a DNA fragment, which encodes the nitrite-eliminating enzyme (NEE) activity. This was achieved by enriching the activity using the fast performance liquid chromatography (FPLC) and protein sequencing. The two internal peptide sequences were obtained: L P G D Y T D Q L L R and S G A I V G L G H A Q V D R. These were used to design the degenerate primers in order to pool a clone from nocardioform genome. Two different genomic libraries were constructed and screened using R. opacus HL PM-1 and N. simplex FJ2-1A total DNA, since the activity in crude extracts was reported with both strains. The partial DNA sequence of NEE was obtained (252 bp). The similarity with fatty acid hydroxylase from Nocardioides sp. JS6614 was detected.

Rhodococcus, a species of bacteria commonly found in the soil, is an under-explored producer of small bioactive compounds including siderophores, pigments and antibiotics. MTM3W5.2 is a strain of Rhodococcus that was previously discovered to produce an antibiotic-like compound that has inhibitory effects on other Rhodococcus strains, including the veterinary pathogen, R. equi. The biosynthetic gene cluster responsible for production of the antibiotic has been identified, and a small gene, BTZ20_3964 at the start of the operon is believed to be a luxR solo regulator of the gene cluster. The goal of this project was to determine this gene’s status as a regulator for the gene cluster. Merodiploids were constructed using the deletion construct, pEX18Km3964AD to obtain a double crossover recombination event to replace the functional gene with the deletion construct. However, evidence indicates that an illegitimate recombination event occurred to produce a merodiploid strain.

Graduation date: 2005