<p>C₄-dicarboxylic acids appear to be metabolized via the TCA cycle in N₂-fixing bacteria (bacteroids) within legume nodules. In <em>Sinorhizobium meliloti</em> bacteroids from alfalfa, NAD⁺-malic enzyme (DME) is required for symbiotic N₂-fixation and this activity is thought to be required for the anaplerotic synthesis of pyruvate. In contrast, in the pea symbiont <em>Rhizobium leguminosarum</em> pyruvate synthesis can occur via either the DME pathway or a pathway catalyzed by phosphoenolpyruvate carboxykinase (PCK), pyruvate kinase (PYK), and pyruvate dehydrogenase. Here we report that <em>dme</em> mutants of <em>Sin</em>or<em>hizobium sp</em>. NGR234 formed root nodules on a broad range of plants and that the level of N₂-fixation varied from 90% to 20% of wild type depending on the host plants inoculated. NGR234 bacteroids had significant PCK activity and while single <em>pckA</em> and single <em>dme</em> mutants fixed N₂ on <em>Macroptilium atropurpureum</em> and <em>Leucaena leucocephala</em> (albeit at a reduced rate), a <em>pckA</em> <em>dme</em> double mutant had no N₂-fixing activity (Fix^-). Thus, NGR234 bacteroids appear to synthesize pyruvate from TCA cycle intermediates via DME or PCK pathways. These NGR234 data, together with other reports, suggested that the completely Fix^- phenotype of <em>S. meliloti dme </em>mutants may be specific to the alfalfa-<em>S. meliloti </em>symbiosis. We therefore examined the ME-like genes <em>azc3656 </em>and <em>azc0119 </em>from <em>Azorhizobium caulinodans</em>, as <em>azc3656 </em>mutants were previously shown to form Fix^- nodules on the tropical legume <em>Sesbania rostrata</em>. We found that purified AZC3656 protein is an NAD (P)⁺-malic enzyme whose activity is inhibited by acetyl-coenzyme A (acetyl-CoA) and stimulated by succinate and fumarate. Thus, whereas DME is required for symbiotic N₂ fixation in <em>A. caulinodans </em>and <em>S. meliloti</em>, in other rhizobia this activity can be bypassed via another pathway(s).</p> <p>In <em>S. meliloti</em> both malic enzymes DME and TME share similar apparent <em>K_m</em>s for substrate and cofactors, but differ in their responses to TCA cycle intermediates, with DME activity inhibited by acetyl-CoA and induced by succinate and fumarate. Previous results in our laboratory indicated that DME is essential for symbiotic N₂ fixation, while TME fails to functionally replace DME. One possible reason for it is that a high ratio of NADPH/NADP⁺in<em> S. meliloti </em>bacteroids prevents TME from functioning in nodules. We sought to lower the<em> </em>NADPH/NADP⁺ratio by overexpressing a soluble pyridine nucleotide transhydrogenase (STH). However, metabolite measurements indicated that overproducing STH failed to lower the ratio of NADPH/NADP⁺ in<em> S. meliloti</em>.</p> <p>Previous studies assumed that DME and TME might play different roles in central carbon metabolism. To gain insight of their physiological functions, genome-wide microarray analysis was conducted in <em>S. meliloti</em> single<em> dme and</em> <em>tme</em> mutants grown on glucose or succinate. The most striking changes of gene expression were observed in <em>S. meliloti</em> <em>dme</em> mutants grown on succinate. The functions of upregulated genes suggested that DME might play an important role in regulating TCA cycle intermediates, important for the maintenance of metabolic flux through TCA cycle during C₄-dicarboxylate oxidation. However, changes of gene expression found in <em>tme </em>mutants were not significant enough to predict the physiological functions of TME protein in central carbon metabolism.</p> / Doctor of Philosophy (PhD)

Due to the recent rise in antibiotic resistant pathogens, and the difficulties surrounding the quest for new antibiotics, many researchers have started revisiting antibiotic interactions in hopes of finding new treatment options. The primary outcome of this project was to examine the efficacy of concomitant antibiotic use under varying nutrient conditions, to identify variations in antibiotic interactions. Antibiotic interactions were studied, utilizing E. coli as a model bacterial system, grown in four different media types. E. coli cultures were treated with streptomycin, tobramycin, erythromycin, and amikacin individually and in a pairwise fashion at varying doses. We found that at least some antibiotic efficacies were dependent on the environmental nutrient conditions E. coli was grown in, as the antibiotics were not equally effective in all media types. E. coli grown in potato dextrose broth, in particular, showed extremely high tolerance to antibiotic inhibition. In addition, we observed several variations in antibiotic interactions, depending on the combination of antibiotics and environmental conditions utilized. It is predicted that differences in available nutrients is the primary cause of the observed discrepancies in antibiotic properties between media. The observation of changes in antibiotic efficacy under different environmental and nutrient conditions has serious implications for use of antibiotic combinations as drug treatments. Not all microenvironments within the human body have identical nutrient make-up. If the interactions antibiotics are reported to have in one environmental condition change under another, reckless prescription of combinations could lead to a serious adverse reaction. Thus, this is an important area for future in vitro and in vivo research.

Dans le cadre de ce travail de thèse, la physiologie apparente de Streptomyces pristinaespiralis et plus spécifiquement la production de pristinamycines (déclenchement et concentration) a été reliée à son environnement hydrodynamique. Des cultures de S. pristinaespiralis ont été réalisées sous diverses conditions d'agitation et d'aération, en fioles lisses d’Erlenmeyer. Ces conditions engendrent des dissipations volumiques comprises entre 0,55 et 14 kW.m-3 et des kLa compris entre 30 et 490 h-1. Partant du constat de la complexité combinée de l’hydrodynamique rencontrée dans les bioréacteurs et de la réponse cellulaire, nous avons développé une approche pluridisciplinaire et multiéchelle à l’interface entre génie des procédés et physiologie quantitative. La réponse physiologique apparente a été quantifiée en termes de croissance, consommation des substrats, morphologie et production. L’hydrodynamique des fioles agitées a été notamment décrite par utilisation de la simulation numérique des écoulements. Par l’utilisation originale d’un modèle de rupture, les diamètres des pelotes ont été corrélés à l’échelle de dissipation de Kolmogorov. De plus, il a été montré que la dissipation défavorisait la croissance des pelotes. Ainsi, par le découplage de l’agitation et de l’aération, il a été montré que la taille des pelotes, contrôlée par la turbulence, impactait directement la consommation d’oxygène et la quantité de pristinamycines produites. Par ailleurs, le déclenchement de la production, résultante d’une limitation en substrats azotés et d’un apport en oxygène suffisant, est déterminé conjointement par la quantité du transfert d’oxygène et par la dissipation volumique / During this study, the physiology of Streptomyces pristinaespiralis and more specifically the production of pristinamycins (induction and concentration) were related to its hydrodynamic environment. Cultures of S. pristinaespiralis were performed under various conditions of agitation and aeration in non baffled Erlenmeyer flasks. According to the operating conditions, the volume power dissipation was from 0.55 to 14 kW.m-3 while kLa was from 30 to 490 h-1. Based on the observation of the complexity of both hydrodynamics encountered in bioreactors and of the cellular response, a multiscale and multidisciplinary approach between process engineering and quantitative physiology was developed. The apparent physiological response was quantified in terms of growth, substrates consumption, morphology and production. The hydrodynamics of the shake flasks was described using Computational Fluid Dynamics. Using an original break up model, the pellet diameters were correlated to the Kolmogorov dissipation scale. Moreover, it was shown that pellet growth was slowed down by the dissipation scale increase. Then, by decoupling the agitation and the aeration, it was shown that the pellets size, controlled by turbulence, impacted directly the consumption of oxygen and the concentration of pristinamycins. Furthermore, onset of pristinamycin production resulted in a limitation in nitrogen substrates as well as a sufficient oxygen supply which are determined by the oxygen transfer and the volume power dissipation

Klebsiella pneumoniae is an opportunistic pathogen responsible for lobar pneumoniae, liver abscess, and septicemia. Clinical isolates are found to be extended spectrum beta lactamase positive with differential expression of the two classical porins, OmpK35 and OmpK36. Porin loss is associated with increased minimum inhibitory concentrations of beta lactam, cephalosporin, and carbapenem antibiotics that target the peptidoglycan. However, little is known about how porin loss affects other aspects of the cell envelope. The focus of this study was to characterize clinical isolates exhibiting differential porin expression and determine if the cumulative changes altered the resistance to phagocytosis by macrophages. The results support the hypothesis that porin loss significantly impacts the overall cell envelope composition, which in turn alters interactions with macrophages.

Ribosomal protein L27 is a component of the eubacterial large ribosomal subunit that has been shown to play a critical role in substrate stabilization during protein synthesis. This function is mediated by the L27 N-terminus, which protrudes into the peptidyl transferase center where it interacts with both A-site and P-site tRNAs as well as with 23S rRNA. We observed that L27 in S. aureus and other Firmicutes is encoded with a short N-terminal extension that is not present in most Gram-negative organisms, and is absent from mature ribosomes. The extension contains a conserved cleavage motif; nine N-terminal amino acids are post-translationally removed from L27 by a site-specific protease so that conserved residues important for tRNA stabilization at the peptidyl transferase center are exposed. We have identified a novel cysteine protease in S. aureus that performs this cleavage. This protease, which we have named Prp, is conserved in all bacteria containing the L27 N-terminal extension. L27 cleavage was shown to be essential in S. aureus; un-cleavable L27 did not complement an L27 deletion. Cleavage appears to play an essential regulatory role, as a variant of L27 lacking the cleavage motif could not complement. Ribosomal biology in eubacteria has largely been studied in E. coli; our findings indicate that there are aspects of the basic biology of the ribosome in S. aureus and other related bacteria that differ substantially from that of E. coli. This research lays the foundation for the development of new therapeutic approaches that target this novel, essential pathway.

More so than ever, Multiple Drug Resistant (MDR) bacteria are on the rise due to overuse of antibiotics along with natural selection for adaptations that enhance drug-resistant properties. One particular bacterial family, Enterobacteriaceae, has been problematic, exhibiting several bacterial members that have developed a precipitous resistance to modern antibiotics and are also primary causative agents of nosocomial, or hospital acquired, infections. Citrobacter freundii (CF) and Enterobacter cloacae (ECL) are two species of the Enterobacteriaceae family causing significant medical concern due to their role in producing numerous opportunistic infections such as bacteremia, lower respiratory tract infections, urinary tract infections, and endocarditis. Adding to the difficulty of this situation is the ability of bacteria to produce biofilms. These biofilms are communities of bacteria that exhibit increased resistance to antibiotic treatment and eradication. Previous work in the laboratory of Dr. Fox at ETSU has identified an uncharacterized product secreted by Klebsiella pneumoniae (KP), another member of the Enterobacteriaceae family, which appears to have inhibitory effects toward CF and ECL. The current study was designed to characterize the effects this secreted product has on CF and ECL biofilms. Through a high throughput microtiter plate assay, the effects of this secreted product were examined on CF and ECL phases of biofilm attachment and maturation. Based on our findings, we have concluded that this secreted product can be categorized as a possible bacteriostatic agent against biofilm cell density, biofilm mass, and cell viability for both biofilm phases of attachment and maturation.

Les communautés microbiennes de sédiments de subsurface ont été décrites jusqu’à 1922 mbsf (meters below the seafloor) et pourraient représenter 0,6% de la biomasse totale. Largement incultivées, ces communautés comprennent des groupes endémiques aux environnements de subsurface et des généralistes retrouvés dans des environnements contrastés, appartenant aux 3 domaines du vivant (Bacteria, Eukarya and Archaea). Bien que jouant un rôle majeur dans les grands cycles géochimiques, l’écologie microbienne des sédiments de subsurface reste peu connue. Les conditions hostiles de ces sédiments contrastent avec la présence d’activité et de viabilité microbiennes. Dans ce contexte, de nombreuses questions sur les modes de vie et les métabolismes des microorganismes enfouis demeurent. L’objectif de cette thèse était de mieux comprendre quelles stratégies adaptatives pouvaient être mises en place par les communautés microbiennes de subsurface et de caractériser leur potentiel physiologique. Pour cela, 3 approches ont été utilisées.(1) Une approche culturale a permis de décrire 2 nouvelles espèces bactériennes sédimentaires (Halomonas lionensis, ungénéraliste versatile, et Phaeobacter leonis, une bactérie marine typique). L’étude de la résistance aux conditions de subsurface de ces deux espèces et de la bactérie Sunxiuqinia faeciviva, isolée à 247 mbsf, a ensuite été étudiée. (2) Par une étude de génomique comparée et structurale, la plasticité physiologique de H. lionensis a été analysée. (3) Enfin, le potentiel fonctionnel de communautés microbiennes enfouies à 31 et 136 mbsf dans le bassin de Canterbury a été étudié, en analysant les 2métagénomes correspondants. Les résultats culturaux et génomiques montrent que H. lionensis et S. faeciviva résistent mieux aux stress de subsurface que P. leonis et, dans le cas de H. lionensis, ceci impliquerait des propriétés physiologiques variées pouvant expliquer le succès écologique du genre Halomonas. Les données de métagénomique indiquent que les diversités phylogénétique et fonctionnelle de subsurface du bassin de Canterbury sont distinctes de celles d’environnements de surface et suggèrent que des métabolismes comme la fermentation, la méthanogenèse ou la β-oxydation pourraient être importants. La présence de gènes d’importance écologique et évolutive a permis d’émettre des hypothèses sur les modes de vie de ces microorganismes et des évènements de recombinaison génomique de groupes toujours incultivés ont aussi pu être décrits / Microbial communities inhabiting marine subsurface sediments were described up to 1922 mbsf (meters below the sea floor) andcould represent 0.6% of the total biomass. This microbial diversity, remaining elusive to cultivation, comprises groups specific to subsurface environments and groups of generalists found in contrasted habitats, all belonging to the 3 domains of life (Bacteria,Eukarya and Archaea). Although playing a major role in global geochemical cycles, the microbial ecology of the subseafloor remains largely unknown. The hostile conditions of subsurface sediments contrast with the descriptions of microbial activity andviability in the subseafloor. In this context, many questions related to the microbial physiology and the lifestyles of buried communities remain to be answered. The objective of this thesis was to better understand which adaptive strategies could be deployed by subseafloor microbial communities and to characterize their physiological potential. In that aim, 3 approaches were used.(1) A cultural approach enabled describing 2 novel sedimentary bacterial species (Halomonas lionensis, a versatile generalist and Phaeobacter leonis a typical marine bacterium). The survival of these 2 species to subseafloor conditions and of the subsurface bacteria Sunxiuqinia faeciviva, isolated at 247 mbsf, was then studied. (2) Using a structural and comparative genomic approach, the physiological plasticity of H. lionensis was investigated. (3) Finally, the functional potential of the microbial communities buried at 31 and 136 mbsf in the Canterbury Basin was analyzed, by studying the 2 corresponding metagenomes. Cultural and genomics results showed that H. lionensis and S. faeciviva are more resistant to subsurface constrains than P. leonis and, in the case of H. lionensis, this may involve various physiological properties, maybe explaining thee cological success of the genus Halomonas. Metagenomic data showed that the functional and the phylogenetic diversity of the subseafloor are distinct from the ones from surface environments and highlighted the importance of metabolic pathways like fermentation, methanogenesis and β-oxidation. Genes of ecological and evolutionary interests enabled speculating about lifestyles of buried microorganisms and analyses of genomic fragments highlighted recombination events of still uncultivated microbial groups

Planktothricoides raciborskii and Roseofilum reptotaenium are physiologically similar, yet ecologically distinct organisms found in a hot spring outflow and coral black band disease (BBD), respectively. The aim of this study was to elucidate the relationship between R. reptotaenium and sulfide in BBD, to compare microcystin (MC) production in response to environmental factors, and to determine chemotactic responses to MC and sulfide by the two organisms. Results showed that the pathogenicity of R. reptotaenium in BBD is dependent on sulfate-reducing bacteria as secondary pathogens. Roseofilum reptotaenium produced significantly more MC than P. raciborskii, as measured using ELISA. Roseofilum reptotaenium was negatively chemotactic to sulfide, determined using horizontal and vertical gradients in agar, while P. raciborskii was not affected. Neither cyanobacterium was chemotactic to MC in the agar assays. The ecophysiology of P. raciborskii and R. reptotaenium in relation to MC production and response to sulfide reflected their pathogenic versus non-pathogenic status.

Quorum sensing molecules have become a recent focus of study to learn if and how they can be used, both on their own and in conjecture with current antimicrobial methods, as a means of bacterial control. One such quorum sensing molecule is the sesquiterpene alcohol, Farnesol, which is synthesized and released by the fungus, Candida albicans. In most in-vivo cases, our laboratory has shown that Alcaligenes faecalis overtakes C. albicans, preventing its growth. However, as a way to counteract this inhibitory effect, Farnesol may be one way that Candida has found to fight back. In this study, we focused on the inhibitory properties of Farnesol for growth and motility of A. faecalis, as well as, the molecule’s ability to prevent Alcaligenes from creating biofilms and/or degrading them once they have already been established. Our experiments show evidence that Farnesol is able to inhibit both the growth and motility of A. faecalis, and determination of the specific concentrations of Farnesol needed to see the largest effects on A. faecalis biofilms. Our hope is that in future studies, we will be able to add varying concentrations of the Farnesol to known and widely used antibiotics in order to increase the effectiveness of antibiotics against bacterial strains, both in the Alcaligenes genus and in other genus, that have previously been considered “antibiotic resistant”.

While the major events in prokaryotic cell cycle progression are likely to be coordinated with transcriptional and metabolic changes, these processes remain poorly characterized. Unlike many rapidly-growing bacteria, DNA replication and cell division are temporally-resolved in mycobacteria, making these slow-growing organisms a potentially useful system to investigate the prokaryotic cell cycle. To determine if cell-cycle dependent gene regulation occurs in mycobacteria, we characterized the temporal changes in the transcriptome of synchronously replicating populations of Mycobacterium tuberculosis (Mtb). By enriching for genes that display a sinusoidal expression pattern, we discover 485 genes that oscillate with a period consistent with the cell cycle. During cytokinesis, the timing of gene induction could be used to predict the timing of gene function, as mRNA abundance was found to correlate with the order in which proteins were recruited to the developing septum. Similarly, the expression pattern of primary metabolic genes could be used to predict the relative importance of these pathways for different cell cycle processes. Pyrimidine synthetic genes peaked during DNA replication and their depletion caused a filamentation phenotype that phenocopied defects in this process. In contrast, the IMP dehydrogenase guaB2 dedicated to guanosine synthesis displayed the opposite expression pattern and its depletion perturbed septation. Together, these data imply obligate coordination between primary metabolism and cell division, and identify periodically regulated genes that can be related to specific cell biological functions.