PhD / Dissertation presented for the degree of Doctor of Philosophy at Stellenbosch University / Embargo(30)lift date 2009-12-31 plt 2010 / ENGLISH ABSTRACT: Through powerful signal molecules, rhizobacteria affect fundamental processes in plants. In recent years, a number of novel rhizobial molecules have been identified that positively affect plant growth and development. Previous studies have shown that Sinorhizobium meliloti, which form symbiotic relationships with leguminous plants, increases CO2 availability by enhancing root respiration in alfalfa. The active compound was identified as lumichrome, a previously unrecognized rhizosphere signal molecule that has been shown to promote plant growth in various studies. Lumichrome is a common breakdown product of riboflavin and produced by both chemical and biological factors. Various studies on lumichrome have proven its growth promoting effect in the interaction with plants. The mechanism through which lumichrome increases plant growth remains to be clarified. This study provides new insight into the molecular effects of the plant growth promoter lumichrome on the root metabolism of plants. The main aim of the work presented in this thesis was to investigate the molecular mechanism of the plant growth promoting substance lumichrome in the roots of the model plants Lotus japonicus and Solanum lycopersicon (tomato). To asses the impact of lumichrome on the root metabolism of Lotus japonicus and tomato and identify key genes involved in the growth stimulation, a comprehensive profile of differentially expressed genes, proteins and metabolites was compiled. As the effects of lumichrome as a plant growth promoter have not previously been tested on Lotus japonicus and tomato, basic growth studies were completed to determine if lumichrome indeed elicits plant growth at nanomolar concentrations, as was proven in numerous previous studies. Both Lotus japonicus and tomato showed significant increases in root biomass when treated with 5 nM of lumichrome. The treatment with lumichrome caused complex changes in gene expression. Generally, transcript profiling showed that the categories that were predominantly affected by lumichrome in both Lotus and tomato, were genes associated with RNA regulation of transcription and signaling, protein synthesis/degradation/modification and stress and defence. Proteomic studies revealed that the majority of the differentially expressed proteins were down-regulated. Lumichrome seems to largely influence proteins involved in protein folding and down-regulate proteins involved in glycolysis. Proteomics studies revealed that GS1 (Lotus) and GAPDH (Lotus and tomato) were present in lower abundance in lumichrome treated roots, therefore targeted analysis utilizing northern blots, western blots and the measurement of enzyme activities were completed to determine and verify their specific role in the lumichrome mediated growth promotion. The results indicated that GAPDH and GS1 seem to be under post-translational modification. The influence of lumichrome on the metabolome of Lotus roots was immense, however minute in tomato roots. The knowledge gained in the parallel analyses of both Lotus japonicus and tomato aided us in finding key genes involved in the growth stimulation. Overall, one of the most significant observations was that for the first time to our knowledge, six genes related to defence and pathogen responses were identified that are concurrently expressed in both Lotus and tomato. Through identifying a small number of genes involved in mediating the growth stimulation, these can be used for their functional analysis in the future, using reverse genetics to provide more insight into the molecular mechanisms that are triggered by lumichrome as a plant growth promoter. / AFRIKAANSE OPSOMMING: Deur kragtige sein-molekules, beïnvloed rhizobakterieë basiese prosesse in plante. In die laaste jare is ʼn aantal nuwe molekules, afkomstig van rhizobakterieë, geidentifiseer wat plantgroei en ontwikkeling positief beïnvloed. Voorafgaande studies het bewys dat Sinorhizobium meliloti, wat simbiotiese verhoudings met peulplante aangaan, die beskikbaarheid van CO2 vermeerder deur wortel respirasie in alfalfa te verhoog. Die aktiewe komponent is as lumikroom geidentifiseer, 'n vroeë onerkenbare risosfeer sein-molekule, wat deur vorige studies bewys is dat dit plantgroei stimuleer. Lumikroom is ʼn algemene afbreekproduk van riboflavin en word geproduseer deur chemiese en biologiese faktore. Verskeie studies op lumikroom het bewys dat dit 'n groei stimuleerende effek het op die groei van plante as dit daarmee in wisselwerking tree. Die meganisme waarmee lumikroom plante groei verhoog, is nog nie opgeklaar nie. Hierdie studie verleen nuwe insigte in die molekulêre effekte van die plantgroei stimuleerende molekuul lumikroom op die wortel metabolisme van plante. Die hoofdoel van die werk wat voorgestel word in hierdie tesis, was om die molekulêre meganisme van die plantgroei stimuleerende stof, genaamd lumikroom, in die wortels van die model plante Lotus japonicus en Solanum lycopersicon (tamatie), te ondersoek. Om die uitwerking van lumikroom op die wortel metabolisme van Lotus japonicus en tamatie te bepaal, asook sleutelgene wat betrokke is by die groei stimulasie te identifiseer, is 'n breedvoerige profiel van differensiële uitgedrukte gene, proteïne en metaboliete saamgestel. Die effekte van lumikroom as 'n plantgroei stimuleerende stof is nog nooit op Lotus japonicus en tamatie getoets nie. Om díe rede is eers basiese plantgroei studies gedoen, om vas te stel of lumikroom inderdaad plantgroei teen nanomolare konsentrasies stimuleer, soos in vele voorafgaande studies bevestig is. Beide Lotus japonicus en tamatie het aansienlike verhogings in wortel biomassa getoon as dit met 5 nM lumikroom behandel is. Die behandeling van plante met lumikroom het komplekse veranderinge in geen-uitdrukking veroorsaak. Oor die algemeen het die transkrip-profiele gewys dat die kategorieë wat die meeste geraak is deur lumikroom behandeling, in beide Lotus en tamatie, gene was wat geassosieer word met RNS regulasie van transkripsie en sein-netwerke, proteïen sintese/degradasie/wysiging en stres en verdedigings prosesse in plante. Proteïen studies het gewys dat daar 'n daling in die meerderheid van die proteïen vlakke was wat differensieël uitgedruk was. Dit blyk dat lumikroom in 'n groot mate proteïene beïnvloed wat betrokke is by proteïen-vouing en veroorsaak dat proteïen vlakke van glikolitiese ensieme daal. Proteïen studies het gewys dat GS1 en GAPDH in laer vlakke teenwoordig was in lumikroom behandelde plante en daarom is 'n meer doelgerigte analiese gedoen deur gebruik te maak van "northern blot", "western blot" en deur die ensiem aktiwiteite te meet om hulle spesifieke rol in die lumikroom bemiddelde groei vas te stel. Die resultate wys daarop dat GAPDH en GS1 mag onder die invloed van na-translasionele verandering wees. Die invloed van lumikroom op die metabolietvlakke was groot in Lotus wortels, maar dit het minder van 'n effek gehad op tamatie wortels. Die kennis wat opgedoen is deur die paralelle analiese van beide Lotus japonicus en tamatie plante help ons om sleutel gene wat betrokke is by groeistimulasie te identifiseer. Een van die betekenisvolste waarnemings van hierdie studie was dat vir die eerste keer, sover ons kennis strek, ses gene wat almal betrekking het tot verdediging en patogene-reaksies, geidentifiseer is wat gelyktydig in beide Lotus en tamatie uitgedruk word. Deur 'n klein aantal gene te identifiseer, wat betrokke is by groeistimulasie, kan die gene in die toekoms vir funksionele analieses gebruik word deur van keerkoppeling-genetika gebruik te maak. Daardeur sal meer insig verkry word in die molekulêre meganisme wat deur lumikroom as 'n plantgroei stof veroorsaak word.

No description available.

No description available.

The effect of ethanol on the stress response in rats was examined. Experimental animals were given 0.25 ml of 28 percent ethanol or 0.25 ml of water orally once a day, five days a week, for a period of twelve months and were then subjected to fifteen minute cold stress. Corticosterone levels in ethanol-treated males following stress were significantly lower (22 percent) than in the sham group. Adrenal weights in sham-treated females were significantly higher (15 percent) than in the ethanol group at the end of twelve months. Mortality in sham-treated males was significantly higher (60 percent) than in ethanol-treated males. The effects observed may be due to the sedative action of ethanol on cortical centers controlling the hypothalmus.

No description available.

Quorum sensing (QS) is a type of cell-to-cell communication that allows the bacteria to communicate via small molecules to coordinate activities such as growth, biofilm formation, virulence, and stress response as a population. QS depends on the accumulation of signal molecules as the bacterial population increases. After a critical threshold of the signal molecules are reached, the bacteria induce a cellular response allowing the bacteria to coordinate their activities as a population. In Vibrio anguillarum, three parallel quorum-sensing phosphorelay systems channels information via three hybrid sensor kinases VanN, VanQ, and CqsS that function as receptors for signal molecules produced by the synthases VanM, VanS, and CqsA, respectively. The phosphorelay systems converge onto a single regulatory pathway via the phosphotransferase VanU, which phosphorylates the response regulator VanO. Together with the alternative sigma factor RpoN, VanO activates the expression of a small RNA, Qrr1 (Quorum regulatory RNA), which in conjunction with the small RNA chaperone Hfq, destabilizes vanT mRNA, which encode the major quorum-sensing regulator in V. anguillarum. This thesis furthers the knowledge on the quorum-sensing phosphorelay systems in V. anguillarum. In this study, three additional qrr genes were identified, which were expressed during late logarithmic growth phase. The signal synthase VanM activated the expression of the Qrr1-4, which stands in contrast to Qrr regulation in other vibrios. Moreover, in addition to VanO, we predict the presence of a second response regulator which can be phosphorylated by VanU and repress Qrr1-4 expression. Thus, VanU functions as a branch point that can regulate the quorum-sensing regulon by activating or repressing VanT expression. Furthermore, VanT was shown to directly activate VanM expression and thus forming a negative regulatory loop, in which VanM represses VanT expression indirectly via Qrr1-4. In addition, VanM expression was negatively regulated post-transcriptionally by Hfq. Furthermore, a universal stress protein UspA repressed VanM expression via the repression of VanT expression. We showed that UspA binds Hfq, thus we suggest that UspA plays a role in sequestering Hfq and indirectly affect gene expression. This thesis also investigated the mechanism by which V. anguillarum can attach to and colonize fish skin tissue. We show unequivocally that fish skin epithelial cells can internalize bacteria, thus keeping the skin clear from pathogens. In turn, V. anguillarum utilized the lipopolysaccharide O-antigen to evade internalization by the fish skin epithelial cells. This study provides new insights into the molecular mechanism by which pathogen interacts with marine animals to cause disease.

Persistent organic pollutants (POPs), including polychlorinated biphenyls (PCBs) are widespread in aquatic systems. These toxicants bioaccumulate in the tissues of aquatic organisms, especially fish as they occupy a position near the top of the aquatic food web. Teleost fish respond to stressors, including toxicants, by activating a co-ordinated network of adaptive responses, collectively termed the integrated stress response, which allows animals to regain homeostasis. Depending on the nature of the stressor, this stress response may be a generalised endocrine response that occurs at the organismal level and/or a cellular response involving protein synthesis. The cellular response to PCB insult involves aryl hydrocarbon receptor (AhR) activation and the induction of biotransformation enzymes, including cytochrome P4501A (Cyp1A). However, little is known about the mode of action of PCBs in affecting the adaptive stress response in animals. The objective of this thesis was to investigate the role played by AhR in mediating PCB impact on the highly conserved physiological responses to secondary stressors in fish. The experimental approach involved whole animal exposure studies with PCBs both in a laboratory setting as well as using feral fish. Also, in vitro mechanistic studies with pharmacological agents [AhR agonist (β-naphthoflavone) and antagonist (resveratrol), Hsp90 inhibitor (geldanamycin), proteasomal inhibitor (MG-132) and transcription (Actinomycin D) and translational inhibitors (cycloheximide D)] were carried out to understand AhR regulation in primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes. Also, a targeted trout cDNA microarray was developed as a tool to identify stress-responsive genes and signaling networks in fish. Short-term (3 day) exposure to PCBs, while inducing liver AhR and Cyp1A expression, did not modify the adaptive plasma cortisol response to an acute handling disturbance in rainbow trout. However, PCBs exposure did modify the metabolic response that is critical for recovery from an acute stressor in rainbow trout. To assess the impact of chronic PCB exposure on cellular stress response, two feral populations of Arctic char (Salvelinus alpinus) from Bjørnøya Island, Norway, were utilized. This is because the average PCB load in char liver from Lake Ellasjøen was approximately 25-fold higher than in individuals from Lake Øyangen, providing a natural setting to compare long-term toxicant impact on stress proteins. Liver Cyp1A expression was elevated in the high PCB fish suggesting AhR activation. Changes in mRNA abundance and/or protein expression of glucocorticoid receptor (GR), heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) in fish from the high PCB lake leads to the proposal that chronic exposures to PCBs is proteotoxic to the fish. In vitro mechanistic studies with trout hepatocytes revealed for the first time that AhR is autoregulated in response to ligand activation in rainbow trout. Furthermore this AhR regulation as well as AhR signaling involves both the molecular chaperone Hsp90 and the proteasome in hepatocytes. AhR signaling appears to play a role in the cellular response to heat shock in trout hepatocytes. Specifically, AhR signaling appears to be involved in the heat shock-induced Hsp70 and Hsp90 protein expression in trout hepatocytes. This modulation of Hsps by AhR may involve the proteasome. Overall, the results point to a cross-talk between the AhR and Hsps signaling pathways, while the precise mechanism(s) remains to be elucidated. A targeted rainbow trout cDNA microarray was constructed as a tool to identify stress-responsive genes in trout. This custom cDNA array consisted of 147 rainbow trout genes designed from conserved regions of fish sequences available in GenBank. The targeted genes had established roles in physiological processes, including stress and immune function, growth and metabolism, ion and osmoregulation and reproduction. This targeted array revealed changes in gene expression suggesting a rapid liver molecular reprogramming as critical for the metabolic adjustments to an acute stressor in fish. Also, transcripts not previously implicated in the stress response process in fish, including genes involved in immune function and protein degradative pathways, were found to be stress-responsive. Many of these transiently elevated stress-responsive transcripts were also shown previously to be glucocorticoid-responsive in fish implicating a key role for genomic cortisol signaling in stress adaptation. Overall, this thesis demonstrates that PCBs impact the organismal and cellular stress response in fish. AhR autoregulation may be a key aspect of PCBs impact on the cellular stress response pathways. Hsp90 and the proteasome may be involved in AhR regulation and PCB-mediated signaling in fish. The results suggest a cross-talk between AhR and Hsp signaling pathways in fish. Finally, the targeted cDNA microarray will be a useful tool to further expand our knowledge on PCBs impact on the cellular stress signaling pathways in fish.

Persistent organic pollutants (POPs), including polychlorinated biphenyls (PCBs) are widespread in aquatic systems. These toxicants bioaccumulate in the tissues of aquatic organisms, especially fish as they occupy a position near the top of the aquatic food web. Teleost fish respond to stressors, including toxicants, by activating a co-ordinated network of adaptive responses, collectively termed the integrated stress response, which allows animals to regain homeostasis. Depending on the nature of the stressor, this stress response may be a generalised endocrine response that occurs at the organismal level and/or a cellular response involving protein synthesis. The cellular response to PCB insult involves aryl hydrocarbon receptor (AhR) activation and the induction of biotransformation enzymes, including cytochrome P4501A (Cyp1A). However, little is known about the mode of action of PCBs in affecting the adaptive stress response in animals. The objective of this thesis was to investigate the role played by AhR in mediating PCB impact on the highly conserved physiological responses to secondary stressors in fish. The experimental approach involved whole animal exposure studies with PCBs both in a laboratory setting as well as using feral fish. Also, in vitro mechanistic studies with pharmacological agents [AhR agonist (β-naphthoflavone) and antagonist (resveratrol), Hsp90 inhibitor (geldanamycin), proteasomal inhibitor (MG-132) and transcription (Actinomycin D) and translational inhibitors (cycloheximide D)] were carried out to understand AhR regulation in primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes. Also, a targeted trout cDNA microarray was developed as a tool to identify stress-responsive genes and signaling networks in fish. Short-term (3 day) exposure to PCBs, while inducing liver AhR and Cyp1A expression, did not modify the adaptive plasma cortisol response to an acute handling disturbance in rainbow trout. However, PCBs exposure did modify the metabolic response that is critical for recovery from an acute stressor in rainbow trout. To assess the impact of chronic PCB exposure on cellular stress response, two feral populations of Arctic char (Salvelinus alpinus) from Bjørnøya Island, Norway, were utilized. This is because the average PCB load in char liver from Lake Ellasjøen was approximately 25-fold higher than in individuals from Lake Øyangen, providing a natural setting to compare long-term toxicant impact on stress proteins. Liver Cyp1A expression was elevated in the high PCB fish suggesting AhR activation. Changes in mRNA abundance and/or protein expression of glucocorticoid receptor (GR), heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) in fish from the high PCB lake leads to the proposal that chronic exposures to PCBs is proteotoxic to the fish. In vitro mechanistic studies with trout hepatocytes revealed for the first time that AhR is autoregulated in response to ligand activation in rainbow trout. Furthermore this AhR regulation as well as AhR signaling involves both the molecular chaperone Hsp90 and the proteasome in hepatocytes. AhR signaling appears to play a role in the cellular response to heat shock in trout hepatocytes. Specifically, AhR signaling appears to be involved in the heat shock-induced Hsp70 and Hsp90 protein expression in trout hepatocytes. This modulation of Hsps by AhR may involve the proteasome. Overall, the results point to a cross-talk between the AhR and Hsps signaling pathways, while the precise mechanism(s) remains to be elucidated. A targeted rainbow trout cDNA microarray was constructed as a tool to identify stress-responsive genes in trout. This custom cDNA array consisted of 147 rainbow trout genes designed from conserved regions of fish sequences available in GenBank. The targeted genes had established roles in physiological processes, including stress and immune function, growth and metabolism, ion and osmoregulation and reproduction. This targeted array revealed changes in gene expression suggesting a rapid liver molecular reprogramming as critical for the metabolic adjustments to an acute stressor in fish. Also, transcripts not previously implicated in the stress response process in fish, including genes involved in immune function and protein degradative pathways, were found to be stress-responsive. Many of these transiently elevated stress-responsive transcripts were also shown previously to be glucocorticoid-responsive in fish implicating a key role for genomic cortisol signaling in stress adaptation. Overall, this thesis demonstrates that PCBs impact the organismal and cellular stress response in fish. AhR autoregulation may be a key aspect of PCBs impact on the cellular stress response pathways. Hsp90 and the proteasome may be involved in AhR regulation and PCB-mediated signaling in fish. The results suggest a cross-talk between AhR and Hsp signaling pathways in fish. Finally, the targeted cDNA microarray will be a useful tool to further expand our knowledge on PCBs impact on the cellular stress signaling pathways in fish.

No description available.

To determine the impact of stress response on enumeration, cell association status and the viability of Escherichia coli DH5, Staphylococcus aureus ATCC 13565 and Listeria monocytogenes CDC 7762 were evaluated using fluorescence microscopy and were compared with the outcomes of traditional plate count and optical density measurements. Fluorescence microscopy revealed that organic acid stress (acetic and lactic, pH 2.7-3.3) induced cell clumping with little loss of viability in Escherichia coli DH5. Significantly lower values for cell enumeration were found for plate counts and OD600 measurement, likely due to cell clumping in response to organic acid stress. Gram-negative bacteria Escherichia coli DH5 showed higher levels of clumping and subsequent resistance against organic acid stress. Increased cell surface hydrophobicity was found in cells that exhibited more evident clumping. However, inorganic acid stress (hydrochloric and sulfuric, pH 3.0-3.3) induced only very low level of clumping in stationary-phase Escherichia coli DH5 and almost no clumping in other cultures. Osmotic stress, heat and cold shock were not found to induce cell clumping. It has been determined that traditional enumeration methods have significantly underestimated the number of viable bacterial cells when organic acid stress is involved. Plate counts and OD600 measurement therefore need to be reassessed as tools for accurate evaluation of pathogens in food industry. / Food Science and Technology