Recent advances in synthetic biology have enabled the construction of non-native metabolic pathways for production of next-generation biofuels in microbes. One such biofuel is the jet-fuel precursor α-pinene, which can be processed into high-energy pinene dimers. However, accumulation of toxic biofuels in the growth medium limits the possible fuel yield. Overexpression of transporter proteins such as efflux pumps can increase tolerance to biofuels by pumping them out of the cell, thus improving fuel yields. However, too many efflux pumps can compromise the cell as well, creating a trade-off between biofuel toxicity and pump toxicity. In this work we improve the conditions of this trade-off in order to increase pinene tolerance in E. coli. We do so by constructing strains incorporating multiple efflux pumps from a variety of organisms and then testing them for tolerance in growth assay experiments. Previous research has suggested that certain combinations of efflux pumps can confer additional tolerance compared to the individual pumps themselves. However, the functional form of the combination of the tolerance provided by each pump and the toxicity due to their simultaneous activity is unknown. Using differential equations, we developed a growth model incorporating the trade-offs between toxicity of α-pinene and efflux pump activity to describe the dynamics of bacterial growth under these conditions. By analyzing biofuel toxicity and the effects of each efflux pump independently through a series of experiments and mathematical models, we propose a functional form for their combined effect on growth rate. We model the mean exponential growth rate as a function of pump induction and biofuel concentration and compare these results to experimental data. We also apply this technique to modeling toxicity of ionic liquids, a class of corrosive salts that has emerged as and effective chemical for pretreatment of biofuel production feedstock. We compare a model for a variety of ionic liquid responsive efflux pump controllers to that of an IPTG inducible controller and show agreement with experimental data, supporting the model's utility to test control schemes before conducting experiments. The overall goal of this project is to use modeling to guide design of tolerance mechanisms to improve overall biofuel yield.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Expression of the mexCD-oprJ multidrug efflux operon is enhanced by the presence of membrane damaging agents [e.g., the biocide chlorhexidine (Chx)] or mutations in the nfxB gene encoding a repressor of efflux gene expression, both dependent on the AlgU envelope stress response sigma factor. Details of mexCD-oprJ regulation are, however, lacking. In examining the mexCD-oprJ locus, a gene, PA4596, encoding a homologue of NfxB (61% identity) was identified downstream of oprJ, a location conserved in all sequenced Pseudomonas aeruginosa isolates and in Pseudomonas putida. Opposite to mexCD-oprJ, PA4596 expression was reduced by Chx exposure, as assessed using RT-PCR; although like mexCD-oprJ, this was AlgU-dependent (i.e., lost in a ΔalgU strain). Deletion of PA4596 had no impact on Chx resistance indicating that it is not required for Chx-inducible mexCD-oprJ expression/ MexCD-OprJ-dependent Chx resistance. In contrast, mexCD-oprJ expression and the attendant multidrug resistance of nfxB deletion mutants were compromised upon deletion of PA4596, indicating that PA4596 plays a positive role in mexCD-oprJ expression in such mutants. Consistent with this, PA4596 expression increased in nfxB deletion and missense mutants in parallel with mexCD-oprJ. Intriguingly, mexCD-oprJ expression and multidrug resistance were observed in a mutant lacking an nfxB mutation (demonstrating an NfxB-like phenotype) and in an nfxB missense mutant and these were not compromised upon deletion of PA4596. Thus, mexCD-oprJ hyperexpression can be both PA4596-dependent and -independent. A bacterial 2-hybrid assay revealed a PA4596-PA4596 interaction, consistent with the protein forming dimers as NfxB has been shown to do. Two-hybrid assays also demonstrated that NfxB and PA4596 interact. While the functional significance of this remains to be elucidated, it is consistent with their common role in regulating mexCD-oprJ expression and is suggestive of a complex and possibly novel regulatory mechanism. These data highlight the complexity of mexCD-oprJ regulation and the apparently multiple pathways to efflux gene expression, suggestive of multiple roles for this efflux system in P. aeruginosa independent of antimicrobial efflux. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-08-18 14:25:18.107

Pseudomonas aeruginosa is a common soil organism as well as an opportunistic human pathogen. Treatment of P. aeruginosa infections is often complicated by innate resistance to a variety of antimicrobials mediated by multidrug efflux systems. The MexAB-OprM efflux system is constitutively expressed in wildtype strains and contributes to innate antimicrobial resistance, while hyperexpression of the system results in acquired high levels of resistance. MexAB-OprM is hyperexpressed in nalC mutants containing mutations in the gene encoding NalC, a repressor of a two-gene operon, PA3720-armR. armR encodes a protein modulator of MexR, a repressor of mexAB-oprM expression. Previous reports showed that genes encoding the MexAB-OprM efflux system are upregulated in response to pentachlorophenol (PCP), a phenolic compound that is a common environmental contaminant. Induction of mexAB-oprM and PA3720-armR by PCP was confirmed using RT-PCR, and MexAB-OprM was shown to be involved in PCP resistance. An electromobility shift assay (EMSA) showed that PCP interacts with NalC, interfering with its binding to the PA3720-armR promoter region and thereby promoting PA3720-armR expression. Nonetheless, the increase in ArmR did not drive mexAB-oprM expression suggesting that PCP induction of this efflux operon occurred via a different mechanism. A direct PCP-MexR interaction could not be demonstrated using an EMSA. PCP exposure did, however, reduce expression of nalD, encoding a second repressor of mexAB-oprM, which might explain the PCP-promoted increase in mexAB-oprM expression. PCP is unlikely to be the intended inducer(s)/substrate(s) for this system but probably resembles these. Several compounds related to PCP were tested for an interaction with NalC but all were negative in EMSAs. Plants produce a variety of phenolic compounds, which are often antimicrobial and, so, root extracts of various plants were tested for an ability to interact with NalC and interfere with promoter binding. Extracts from Boehmeria tricuspis, Uncaria tomentosa and Ixiolirion tataricum were shown to interact with NalC, suggesting that plant compounds may be the intended inducers/substrates for NalC/MexAB-OprM. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2010-09-10 10:35:16.271

Pseudomonas aeruginosa is resistant to many clinically-relevant antibiotics and this is partly attributable to multidrug efflux systems in this organism. One of these, MexEF-OprN, exports chloramphenicol, fluoroquinolones and trimethoprim and is positively regulated by MexT, encoded by the mexT gene that is located upstream of the efflux genes. MexT also positively regulates the mexS gene that encodes an oxidoreductase of unknown function and whose loss increases expression of mexEF-oprN. A null mutation in the mexS gene of P. aeruginosa increased expression of two 3-gene operons, PA4354-PA4355-PA4356 and PA2813-2812-2811. This increased expression, which paralleled an increase in mexEF-oprN expression in the same mutant, was, like mexEF-oprN, MexT-dependent. The PA4356 (xenB) gene product is homologous to a xenobiotic reductase in P. fluorescens shown to remove nitro groups from trinitrotoluene and nitroglycerin. As nitration is a well-known result of nitrosative stress, a role for xenB (and the co-regulated mexEF-oprN and PA2813-2812-2811) in a nitrosative stress response was hypothesized and tested. Using S-nitrosoglutathione (GSNO) as a source of nitrosative stress, expression of mexEF-oprN, xenB and PA2811 was shown to be GSNO-inducible, though in the case of xenB and PA2811 this was only seen in a mutant lacking MexEF-OprN and only for xenB, this GSNO-inducible expression was dependent upon MexT. Consistent with MexT being required for mexEF-oprN and PA4354-PA4355-PA4356 expression, MexT bound to their promoter regions. Chloramphenicol, a nitroaromatic antimicrobial that is a substrate for MexEF-OprN and resembles a nitrosated nitrosative stress product accommodated by this efflux system induced expression of mexEF-oprN, but not xenB, or PA2811, and this was dependent upon the MexT regulator. In addition to MexT positive-regulating activity of genes in response to nitrosative stress, MexT also negatively regulates the expression of mexAB-oprM through direct binding to its promoter region and the oprD gene, encoding an outer membrane porin that provides a portal of entry for basic amino acids and carbapenem, whose expression was reduced only by GSNO. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2011-05-15 20:59:57.018

The influence water levels have on CO2 and CH4 efflux were investigated at the Loxahatchee Impoundment Landscape Assessment (LILA) research facility, located in Boynton Beach, FL, USA. Measurements of CO2 efflux were taken for 24 h periods four times for one year from study plots. Laboratory incubations of intact soil cores were sampled for CO2, CH4, and redox potential. Additionally, soil cores from wet and dry condition were incubated for determination of enzyme activity and macronutrient limitation on decomposition of organic matter from study soils. Water levels had a significant negative influence on CO2 efflux and redox, but did not significantly influence CH4 efflux. Study plots were significantly different in CH4 efflux and redox potential. Labile carbon was more limiting to potential CO2 and CH4 production than phosphorus, with the effect significantly greater from dry conditions soils. Enzyme activity results were variable with greater macronutrient responses from dry condition soils.

<p><i>Mycobacterium abscessus</i> is an emerging infectious pathogen capable of causing pulmonary disease similar to tuberculosis, but has many intrinsic and extrinsic properties making it more drug-resistant than <i>Mycobacterium tuberculosis</i>. Current treatments, including those used for <i>M. tuberculosis</i> infection, have had poor results. Although <i>in vitro</i> studies have shown promise with drug treatment for this microorganism, clinical trials have been mostly unsuccessful. An <i>in vitro </i>model that mimics the physiological stresses encountered within the human body is likely to enable the discovery of mechanisms of antibiotic resistance used by <i>M. abscessus</i> during infection. Therefore, we subjected <i>M. abscessus</i> to a combination of stresses thought to be encountered by mycobacteria inside the human body. We subjected the pathogen to low oxygen, low pH, and nutrient starvation. This is the first report on subjecting <i>M. abscessus</i> to such a combination of stresses. It is also the first to investigate the effect of the combination of stresses on the tolerance of the pathogen to antibiotics, and the effect of efflux pump inhibitors under such conditions. We found that under these conditions, <i>M. abscessus</i> entered a non-replicating state. We investigated whether the multiple-stressed <i>M. abscessus</i> displayed altered tolerance to antibiotics commonly used to treat infection, and whether efflux pump inhibitors affected the antibiotic resistance under such conditions.<i> </i>We found that when subjected to our multiple stress model, <i>M. abscessus</i> in the reactivating phase had higher tolerance to erythromycin in combination with efflux pump inhibitors verapamil and reserpine compared to non-replicating <i>M. abscessus</i>. Reactivating phase cells had a higher tolerance to antibiotic erythromycin than non-replicating cells. Reactivating phase cells also showed antibiotic tolerance in the presence of ATP. This physiologically-relevant experimental model for <i>M. abscessus</i> could potentially be used in discovering the mechanisms of antibiotic resistance in the pathogen.</p>

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Les systèmes de sécrétion de NO3– de la membrane plasmique des cellules végétales jouent un rôle important dans l'activité stomatique et la réponse des plantes à des stress biotiques et abiotiques. Malgré quelques avancées récentes, ces systèmes restent mal connus sur le plan moléculaire. Mon travail de thèse a consisté à caractériser le rôle physiologique de NAXT2, un membre du sous-groupe NAXT (NitrAte eXcretion Transporter) de la famille des transporteurs NRT1/PTR chez Arabidopsis thaliana. Mes résultats montrent que NAXT2 est un transporteur de NO3– et qu'il est principalement exprimé dans les cellules du péricycle de la racine, au voisinage des vaisseaux xylémiens. Sous contrainte saline et comparativement aux plantes sauvages (WT), un mutant knock-out pour NAXT2 (naxt2-1) présente un défaut de distribution du NO3– vers les feuilles et de sécrétion du NO3– dans la sève xylémienne, qui se traduisent par une diminution des teneurs en NO3– foliaires. NAXT2 est donc impliqué dans la charge du xylème en NO3– sous contrainte saline. Aucune des différences phénotypiques mises en évidence entre le mutant et WT sous contrainte saline n'a été observée sous stress osmotique ou en condition standard, suggérant que NAXT2 est essentiellement impliqué dans la réponse à la composante ionique du stress salin. Enfin, après un traitement salin prolongé, la biomasse foliaire de naxt2-1 est inférieure à celle de WT, ce qui indique que NAXT2 joue un rôle important dans l'adaptation des plantes aux contraintes salines modérées. Dans l'ensemble, ce travail suggère que NAXT2 est impliqué dans une fonction physiologique majeure, la translocation du NO3–, point de contrôle de la distribution du nitrate, et dans l'adaptation de la plante aux contraintes salines. / NO3– secretion systems at the plasma membrane of plant cells play an important role in stomata activity and plant response to biotic and abiotic stresses. Despite of few recent advances, these systems are still poorly known at the molecular level. During my thesis, I worked on the characterization of the physiological role of NAXT2, a member of the NAXT (NitrAte eXcretion Transporter) sub-group from the large NRT1/PTR transporters family in Arabidopsis thaliana. The results presented here show that NAXT2 is a NO3– transporter and that it is mainly expressed in root pericycle cells, close to the xylem vessels. Under salinity constraint and relatively to wild type plants (WT), a NAXT2 knock-out mutant (naxt2-1) displayed a defect in NO3– distribution towards the shoots and in NO3– secretion into the xylem sap, which lead to a decrease in shoot NO3– content. Thus, NAXT2 is involved in NO3– xylem loading under salinity constraint. None of the phenotypic differences described in this work between WT and mutant was observed under osmotic stress or standard culture conditions, suggesting that NAXT2 is specifically involved in response to the ionic component of salt stress. Finally, after a prolonged salt treatment, naxt2-1 shoot biomass was lower than that of WT, indicating that NAXT2 plays an important role in plant adaptation to mild salinity constraint. Altogether, this work suggests that NAXT2 is involved in a major physiological function, the NO3– translocation, control point of nitrate distribution and in plant adaptation to salinity constraint.