Thesis addresses the physiological role of formyl tetrahydrofolate synthetase (Fhs) and bifunctional folate dehydrogenase (FolD) in folate mediated one carbon metabolism in bacteria. Thesis consists of 5 chapters. First chapter provides the details of the literature on folate metabolism, enzymes involved the synthesis and physiological roles various folate co-factors. Second chapter discusses the study of Clostridium perfringens Fhs generation of folD deletion in the support of fhs. Third chapter explores the characterization of the folD deletion strain. Fourth chapter presents the characterization of monofunctional versions of FolD from Clostridium perfringens. Fifth chapters talks about anti-correlation existence of Fhs and PurT (phosphoribosyl glycinamide formyl transferase II) The detailed experimental study is discussed below i. Characterization of Clostridium perfringens Formyl Tetrahydrofolate Synthetase (Fhs) In this chapter we have characterized Fhs from pathogenic Clostridium perfringens. Fhs catalyzes the formation of N10-formyl THF from THF and formate. Previously Fhs has been characterized from various non-pathogenic species of Clostridium. In addition, the detailed kinetic parameters are not known. In this report we have characterized the Fhs Clostridium perfringens and detailed kinetic parameters were determined. We have also shown the biological function by rescue of UV photorepair sensitive strain. ii. One-carbon metabolic pathway rewiring in Escherichia coli reveals an evolutionary advantage of 10-formyltetrahydrofolate synthetase (Fhs) in survival under hypoxia In cells, N10-formyltetrahydrofolate (N10-formyl THF) required for formylation of eubacterial/organeller initiator tRNA and purine biosynthesis is produced by methylene- tetrahydrofolate dehydrogenase/cyclohydrolase (FolD) and/or 10-formyltetrahydrofolate synthetase (Fhs). folD is present in all organisms, where as fhs shows mixed distribution. We show that in E. coli, which naturally lacks fhs, essential function of folD could be replaced with fhs of Clostridium perfringens when provided on a medium copy plasmid or integrated as single copy gene in the chromosome of the ∆folD strains, for their growth in a complex medium. However, these strains require purines and glycine as supplements for growth in M9 minimal medium. The in vivo levels of N10-formyl THF in the ∆folD strains (harboring fhs) were limiting despite their high enzymatic capacity to synthesize the same. Auxotrophy for purines could be alleviated by adding formate to the medium, and that for glycine by engineering THF import into the cells. The ∆folD strains showed high NADP+/NADPH ratio and were hypersensitive to trimethoprim (TMP). Further, the presence of fhs was disadvantageous to E. coli under aerobic growth. However, under hypoxia, E. coli strains harboring fhs outcompeted those lacking it. And, the computational analysis revealed a predominant natural occurrence of fhs in anaerobic and facultative anaerobic bacteria. We also propose that inhibitors aimed at folD could potentiate the effect TMP drugs. iii. 5, 10-methylene-THF dehydrogenase (DH) and 5, 10-methenyl-THF cyclohydrolase (CH) activities of FolD are essential to maintain folate homeostasis and anti-folate resistance While E. coli and many other organisms have folD alone or folD and fhs, Clostridium species possess an annotated bi-functional FolD and an annotated methenyl tetrahydrofolate cyclohydrolase (FchA). Simultaneous presence of 3 enzymes for the synthesis of N10-formyl THF was intriguing. To understand this unusual feature we have cloned Clostridium perfringens CpeFolD and CpeFchA, over expressed and purified to near homogeneity. Biochemical analyses revealed that CpeFolD possess only dehydrogenase activity as opposed to in silico prediction, while CpeFchA possess cyclohydrolase activity as expected. We also show that expression of both proteins together allowed folD deletion in E. coli. From this study we found that presence of dehydrogenase and cyclohydrolase functions are very important in the maintenance of folate homeostasis and anti-folate resistance. iv. Analysis of distribution of fhs and purT genes in the organisms While analysing distribution of fhs across genomes, serendipitously we also found that large number of organism which have fhs lack purT(phosphoribosyl glycinamide formyl transferase II), in short where ever purT was present fhs was absent. This kind of anti-correlation was strictly conserved in Bacillus genes as well. Growth competition experiments were done to address anti-correlation between fhs and purT. Growth competition experiments revealed that simultaneous presence of both purT and fhs is disadvantageous, when compared to presence of either one gene.

A thesis submitted to the Faculty of Engineering and the Built Environment, University of the Witwatersrand, Johannesburg, Republic of South Africa, in fulfillment of the requirements for the degree of Doctor of Philosophy in Engineering. Johannesburg, 2016 / The process of photosynthesis is of great importance as it is the reaction of carbon dioxide (CO2) and water with the help of light, ’free’ energy from the sun, to form useful carbohydrates and oxygen. Photosynthesis is therefore useful both in carbon dioxide mitigation and growing bio-feedstocks towards making biofuel. This thesis aims to address two areas for analysing the photosynthesis process: 1. Looking at the physical limits of the growth; and 2. Improving the production rate of some aquatic plants, such as duckweed and microalgae. To address the first aim, the fundamental concepts of thermodynamics were used to analyse the photosynthetic process. It was found that the theoretical minimum number of moles of photons (NP) required (9–17) is less than the values reported by other researchers, suggesting that the photosynthesis process is highly irreversible and inefficient (operating at 35% efficiency or less). This is because the number of moles of photons will increase with greater process irreversibility (when the entropy generated is greater than zero). If the photosynthesis process is indeed that irreversible then the removal of heat (the heat not used by other cellular processes) by the plant becomes a major problem. It is suggested that transpiration, and other cellular processes, are the processes by which that is done, and it is shown that the water needs of the plant for transpiration would dwarf those needed for photosynthesis. Knowing the fundamental limits to growth could also be of use because if an organism was growing at a rate close to this value there would be no advantage to try to do genetic modification to improve its rate. Following the ideas presented above a spectrophotometer was used not only to obtain the absorption spectrum of algae, but it was also used to grow small samples at specific light wavelengths. The algae species researched was Desmodesmus spp., which, for example, is used to remediate waste water or as a source of feedstock for biofuel production. It also tolerates high CO2 concentrations. This simple experimental method demonstrated that a specific light wavelength (in particular the Secomam Prim spectrophotometer) 440 nm was preferred for the algae growth. It was recommended that this specific light wavelength would be best for growth. It might also be useful to know this fact particularly when designing photobioreactors, as this could reduce the amount of heat released into the surroundings and thus make the process more energy efficient. Interestingly, the wavelength for maximum growth corresponded to one of the peaks in the absorption spectra but there was no increase in growth rate corresponding to any of the other peaks. To address the second aim, the author determined how well predictions on improving the growth of algae (Desmodesmus spp. for example), based on a theoretical model, would work when tested experimentally. What the researcher found was that the method improved algae production, using the same set of equipment. The production was improved by a factor of 1.28 and 1.26 (at product concentrations 1000 mg/L and 600 mg/L respectively) when retaining 40% of the algae suspension. The method may be particularly useful when large amounts of biomass are required as there is no extra cost of purchasing additional equipment. The same model was applied to a growth profile of duckweed (Spirodela polyrhiza 8483, which is convertible into biofuel or a source of food), and the author showed that the model could work if the duckweed was provided with an added carbon source. In order to find an economical and reliable alternative to bridge the scale gap between laboratory and industrial production, the author checked if duckweed species (Spirodela polyrhiza 8483, Spirodela polyrhiza 9509, Lemna gibba 8428, Lemna minor DWC 112, Wolffia cylindracea 7340 and Wolffia globosa 9527) could be cultivated in media less expensive than the basal laboratory medium (Schenk and Hildebrandt). The author found that duckweed can be cultivated more efficiently, and in a more cost-effective manner, in the alternative media types, while maintaining growth rates, RGR 0.09 day-1, and starch contents, 5– 17%(w/w), comparable with that obtained with the conventional laboratory media. Thus, by looking at the photosynthesis process thermodynamically and experimentally, it is shown to be possible to improve the process by using concepts presented in this thesis. / MT2017

Staphylococcus aureus un pathogène opportuniste de l’homme responsable de nombreuses maladies. Son pouvoir pathogène est dû à l’expression de nombreux facteurs de virulence, aussi qu’à sa capacité de s’adapter à son environnement. En pénétrant dans nos tissus S. aureus doit, pour survivre, faire face aux changements environnementaux et à la disponibilité des nutriments. L’expression des gènes impliqués dans ces réponses adaptatives, est soumise à une régulation fine, apportée par les systèmes à deux composants, les facteurs de transcription et les sARN (small ARN). Dans cette étude, j’ai identifié les fonctions d’un sARN, appelé RsaI, qui est réprimé en présence de glucose extérieur. RsaI, réprime la traduction, de plusieurs ARNm impliqués dans le métabolisme carboné et également de IcaR, impliqué dans la synthèse de biofilms. RsaI participe à l’inhibition de plusieurs enzymes de la voie de synthèse des pentoses phosphates et interagit également avec d’autre sARN. Cet ARN multifonctionnel est un véritable senseur du taux de glucose extérieur engendrant ainsi un switch métabolique, nécessaire à la réponse adaptative de S. aureus en conditions infectieuses. / Staphylococcus aureus is a human opportunist pathogenic bacterium capable to colonize different host tissues and organs and therefore generates multiple infectious conditions. Its pathogenic power is due to the expression of multiple virulence factors, and by it’s ability to adapt to the environment. Once entered in human tissues, S. aureus must face environmental changes, as the availability of nutriments to survive. Gene expressions implicated in these adaptive responses are submitted to a fine regulation, carried by two component systems, transcriptional factors, and sRNA (small RNA). In this study, I have identified the functions of a sRNA, called RsaI, which is repressed when the external concentration of glucose is at high levels. RsaI represses the translation of multiple mRNA implicated in the carbon metabolism, including a major glucose transporter, and IcaR, implicated in the biofilms synthesis. Furthermore, RsaI interacts with other sRNA. This multifunctional RNA is a real sensor of the external glucose levels, generating a metabolic switch that is necessary to ensure S. aureus adaptive response in infectious conditions.

Pregnancy complications associated with placental development affect approximately one third of all human pregnancies. Genome health is essential for placental and fetal development, as DNA damage can lead to pregnancy loss and developmental defects. During this developmental phase rapid DNA replication provides an increased opportunity for genome and epigenome damage to occur[1]. Maternal nutrition is one of the principal environmental factors supporting the high rate of cell proliferation and differentiation. Folate functions in one-carbon metabolism and regulates DNA synthesis, DNA repair and gene expression[1]. Deficiencies or defects in gene-nutrient interactions associated with one-carbon metabolism can lead to inhibition of cell division, cell cycle delay and an excessive apoptotic or necrotic cell death rate [2], which may affect placentation. This study is the first to investigate the association between genomic damage biomarkers in late pregnancy complications associated with uteroplacental insufficiency (UPI) including preeclampsia and intrauterine growth restriction (IUGR). The results indicate that genome damage in the form of micronucleated cells in peripheral blood lymphocytes at 20 weeks gestation is significantly increased in women at risk of developing an adverse pregnancy outcome. The observed OR for the high micronuclei frequency may be the highest observed for any biomarker selected in relation to risk of pregnancy complications to date (15.6 – 33.0). In addition, reduced apoptosis was observed in association with increased micronuclei, suggesting that the cells may have escaped specific cell-cycle checkpoints allowing a cell with DNA damage to proceed through mitosis. This study demonstrated that an increase in plasma homocysteine concentration at 20 weeks gestation is associated prospectively with the subsequent development of UPI, indicating a causal relationship. The MTR 2756 GG genotype was significantly associated with increased plasma homocysteine concentration and UPI. Furthermore, the MTHFD1 1958 single nucleotide polymorphism was associated with increased risk for IUGR. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1309296 / Thesis (Ph.D.) -- School of Paediatrics and Reproductive Health, 2007

Metabolismus in Pflanzenzellen ist stark kompartimentiert. Viele Stoffwechselwege haben Reaktionen in mehr als einem Kompartiment. Zum Beispiel wird während der Photosynthese in pflanzlichen Mesophyllzellen Kohlenstoff in Form von Stärke in den Chloroplasten synthetisiert, während es im Zytosol in Form von Sacharose gebildet und in der Vakuole gespeichert wird. Diese Reaktionen sind strikt reguliert um ein Gleichgewicht der Kohlenstoffpools der verschiedenen Kompartimente aufrecht zu erhalten und die Energieversorgung aller Teile der Zelle für anabolische Reaktionen sicher zu stellen. Ich wende eine Methode an, bei der die Zellen unter nicht-wässrigen Bedingungen fraktioniert werden und daher der metabolische Status der während der Ernte herrschte über den ganzen Zeitraum der Auftrennung beibehalten wird. Durch die Kombination von nichtwässriger Fraktionierung und verschiedener Massenspektrometrietechniken (Flüssigchromotagraphie- und Gaschromotagraphie basierende Massenspekrometrie) ist es möglich die intrazelluläre Verteilung der meisten Intermediate des photosynthetischen Kohlenstoffstoffwechsels und der Produkte der nachgelagerten metabolischen Reaktionen zu bestimmen. Das Wissen über die in vivo Konzentrationen dieser Metabolite wurde genutzt um die Änderung der freien Gibbs Energie in vivo zu bestimmen. Mit Hilfe dessen kann bestimmt werden, welche Reaktion sich in einem Gleichgewichtszustand befinden und welche davon entfernt sind. Die Konzentration der Enzyme und der Km Werte wurden mit den Konzentrationen der Metabolite in vivo verglichen, um festzustellen, welche Enzyme substratlimitiert sind und somit sensitiv gegenüber Änderungen der Substratkonzentration sind. Verschiedene Intermediate des Calvin-Benson Zyklus sind gleichzeitig Substrate für andere Stoffwechselwege, als da wären Dihyroxyaceton-phosphat (DHAP, Saccharosesynthese), Fructose 6-phosphat (Fru6P, Stärkesynthese), Erythrose 4-phosphat (E4P, Shikimat Stoffwechselweg) und Ribose 5-phosphat (R5P, Nukleotidbiosynthese). Die Enzyme, die diese Intermediate verstoffwechseln, liegen an den Abzweigungspunkten zu diesen Stoffwechselwegen. Diese sind Trisose phosphat isomerase (DHAP), Transketolase (E4P), Sedoheptulose-1,7 biphosphat aldolase (E4P) und Ribose-5-phosphat isomerase (R5P), welche nicht mit ihren Substraten gesättigt sind, da die jeweilige Substratkonzentration geringer als der zugehörige Km Wert ist. Für metabolische Kontrolle bedeutet dies, dass diese Schritte am sensitivsten gegenüber Änderungen der Substratkonzentrationen sind. Im Gegensatz dazu sind die regulierten irreversiblen Schritte von Fructose-1,6.biphosphatase und Sedoheptulose-1,7-biphosphatase relativ insensitiv gegenüber Änderungen der Substratkonzentration. Für den Stoffwechselweg der Saccharosesynthese konnte gezeigt werden, dass die zytosolische Aldolase eine geringer Bindeseitenkonzentration als Substratkonzentration (DHAP) aufweist, und dass die Konzentration von Saccharose-6-phosphat geringer als der Km Wert des synthetisierenden Enzyms Saccharose-phosphatase ist. Sowohl die Saccharose-phosphat-synthase, also auch die Saccharose-phosphatase sind in vivo weit von einem Gleichgewichtszustand entfernt. In Wildtyp Arabidopsis thaliana Columbia-0 Blättern wurde der gesamte Pool von ADPGlc im Chloroplasten gefunden. Das Enzyme ADPGlc pyrophosphorylase ist im Chloroplasten lokalisiert und synthetisiert ADPGlc aus ATP und Glc1P. Dieses Verteilungsmuster spricht eindeutig gegen die Hypothese von Pozueta-Romero und Kollegen, dass ADPGlc im Zytosol durch ADP vermittelte Spaltung von Saccharose durch die Saccharose Synthase erzeugt wird. Basierend auf dieser Beobachtung und anderen veröffentlichten Ergebnissen wurde geschlußfolgert, dass der generell akzeptierte Stoffwechselweg der Stärkesynthese durch ADPGlc Produktion via ADPGlc pyrophosphorylase in den Chloroplasten korrekt ist, und die Hypothese des alternativen Stoffwechselweges unhaltbar ist. Innerhalb des Stoffwechselweges der Saccharosesynthsese wurde festgestellt, dass die Konzentration von ADPGlc geringer als der Km Wert des Stärkesynthase ist, was darauf hindeutet, dass das Enzym substratlimitiert ist. Eine generelle Beobachtung ist, dass viele Enzmye des Calvin-Benson Zyklus ähnliche Bindeseitenkonzentrationen wie Metabolitkonzentrationen aufweisen, wohingegen in den Synthesewegen von Saccharose und Stärke die Bindeseitenkonzentrationen der Enzyme viel geringer als die Metabolitkonzentrationen sind. / Metabolism in plant cells is highly compartmented, with many pathways involving reactions in more than one compartment. For example, during photosynthesis in leaf mesophyll cells, primary carbon fixation and starch synthesis take place in the chloroplast, whereas sucrose is synthesized in the cytosol and stored in the vacuole. These reactions are tightly regulated to keep a fine balance between the carbon pools of the different compartments and to fulfil the energy needs of the organelles. I applied a technique which fractionates the cells under non-aqueous conditions, whereby the metabolic state is frozen at the time of harvest and held in stasis throughout the fractionation procedure. With the combination of non-aqueous fractionation and mass spectrometry based metabolite measurements (LC-MS/MS, GC-MS) it was possible to investigate the intracellular distributions of the intermediates of photosynthetic carbon metabolism and its products in subsequent metabolic reactions. With the knowledge about the in vivo concentrations of these metabolites under steady state photosynthesis conditions it was possible to calculate the mass action ratio and change in Gibbs free energy in vivo for each reaction in the pathway, to determine which reactions are near equilibrium and which are far removed from equilibrium. The Km value and concentration of each enzyme were compared with the concentrations of its substrates in vivo to assess which reactions are substrate limited and so sensitive to changes in substrate concentration. Several intermediates of the Calvin-Benson cycle are substrates for other pathways, including dihydroxyacetone-phosphate (DHAP,sucrose synthesis), fructose 6-phosphate (Fru6P, starch synthesis), erythrose 4-phosphate (E4P,shikimate pathway) and ribose 5-phosphate (R5P, nucleotide synthesis). Several of the enzymes that metabolise these intermediates, and so lie at branch points in the pathway, are triose-phosphate isomerase (DHAP), transketolase (E4P, Fru6P), sedoheptulose-1,7-bisphosphate aldolase (E4P) and ribose-5-phosphate isomerase (R5P) are not saturated with their respective substrate as the metabolite concentration is lower than the respective Km value. In terms of metabolic control these are the steps that are most sensitive to changes in substrate availability, while the regulated irreversible reactions of fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase are relatively insensitive to changes in the concentrations of their substrates. In the pathway of sucrose synthesis it was shown that the concentration of the catalytic binding site of the cytosolic aldolase is lower than the substrate concentration of DHAP, and that the concentration of Suc6P is lower than the Km of sucrose-phosphatase for this substrate. Both the sucrose-phosphate synthase and sucrose-phosphatase reactions are far removed from equilibrium in vivo. In wild type A. thaliana Columbia-0 leaves, all of the ADPGlc was found to be localised in the chloroplasts. ADPglucose pyrophosphorylase is localised to the chloroplast and synthesises ADPGlc from ATP and Glc1P. This distribution argues strongly against the hypothesis proposed by Pozueta-Romero and colleagues that ADPGlc for starch synthesis is produced in the cytosol via ADP-mediated cleavage of sucrose by sucrose synthase. Based on this observation and other published data it was concluded that the generally accepted pathway of starch synthesis from ADPGlc produced by ADPglucose pyrophosphorylase in the chloroplasts is correct, and that the alternative pathway is untenable. Within the pathway of starch synthesis the concentration of ADPGlc was found to be well below the Km value of starch synthase for ADPGlc, indicating that the enzyme is substrate limited. A general finding in the comparison of the Calvin-Benson cycle with the synthesis pathways of sucrose and starch is that many enzymes in the Calvin Benson cycle have active binding site concentrations that are close to the metabolite concentrations, while for nearly all enzymes in the synthesis pathways the active binding site concentrations are much lower than the metabolite concentrations.

Most members of the Rhizobiaceae possess single copies of the poly-3-hydroxybutyrate biosynthesis genes, phbA, phbB and phbC. Analysis of the genome sequence of Bradyrhizobium japonicum reveals the presence of five homologues of the PHB synthase gene phbC as well as two homologues of the biosynthesis operon, phbAB. The presence of multiple, seemingly redundant homologues may suggest a functional importance. Each B. japonicum phbC gene was cloned and used to complement the pleiotropic phenotype of a Sinorhizobium meliloti phbC mutant; this mutant is unable to synthesize PHB, grow on certain PHB cycle intermediates and forms non-mucoid colonies on yeast mannitol medium. Two of the five putative B. japonicum phbC genes were found to complement the S. meliloti phbC mutant phenotype on D-3-hydroxybutyrate although none of them could fully complement the phenotype on acetoacetate. Both complementing genes were also able to restore PHB accumulation and formation of mucoid colonies on yeast mannitol agar to phbC mutants. In-frame deletions were constructed in three of the five phbC open reading frames in B. japonicum, as well as in both phbAB operons, by allelic replacement. One of the phbC mutants was unable to synthesize PHB under free-living conditions; one of the two phbAB operons was shown to be necessary and sufficient for PHB production under free-living conditions. These mutants also demonstrated an exopolysaccharide phenotype that was comparable to S meliloti PHB synthesis mutants. These strains were non-mucoid when grown under PHB-inducing conditions and, in contrast to wild-type B. japonicum, formed a compact pellet upon centrifugation. Interestingly, none of the mutants exhibited carbon-utilization phenotypes similar to those exhibited by S. meliloti PHB mutants. Wild-type B. japonicum accumulates PHB during symbiosis, and plants inoculated with the phbC mutants demonstrate a reproducible reduction in shoot dry mass. Analysis of bacteroid PHB accumulation in the mutant strains suggests that the phbAB operons of B. japonicum are differently regulated relative to growth under free-living conditions; mutants of the second phbAB operon demonstrated a significant reduction in PHB accumulation during symbiosis. These data suggest that the first phbAB operon is required for PHB synthesis only under free-living conditions, but is able to partially substitute for the second operon during symbiosis. Deletion of both phbAB operons completely abolished PHB synthesis in bacteroids. Analysis of the upstream regions of these genes suggest the existence of putative RpoN binding sites, perhaps indicating a potential mode of regulation and highlighting the metabolic complexity that is characteristic of the Rhizobiaceae. PHB metabolism in S. meliloti has been studied in considerable detail with two notable exceptions. No reports of the construction of either a β-ketothiolase (phbA) or a PHB depolymerase (phaZ ) mutant have ever been documented. The phaZ gene, encoding the first enzyme of the catabolic half of the PHB cycle in S. meliloti, was identified and a phaZ mutant strain was generated by insertion mutagenesis. The phaZ mutant demonstrates a Fix+ symbiotic phenotype and, unlike other PHB cycle mutants, does not demonstrate reduced rhizosphere competitiveness. Bacteroids of this strain were shown to accumulate PHB, demonstrating for the first time that S. meliloti is able to synthesize and accumulate PHB during symbiosis. Interestingly, there is no significant difference in shoot dry mass of plants inoculated with the phaZ mutant, suggesting that PHB accumulation does not occur at the expense of nitrogen fixation. The phaZ mutant strain was also used to demonstrate roles for PhaZ in the control of PHB accumulation and exopolysaccharide production. When grown on high-carbon media, this mutant demonstrates a mucoid phenotype characteristic of exopolysaccharide production. Subsequent analyses of a phoA::exoF fusion confirmed elevated transcription levels in the phaZ mutant background. In contrast, mutants of the PHB biosynthesis gene, phbC, have a characteristically dry phenotype and demonstrate reduced exoF transcriptional activity. The phaZ mutant also demonstrates a significant increase in PHB accumulation relative to the wild-type strain. Previous work on phasin mutants in S. meliloti demonstrated that they lack the ability to synthesize PHB. Transduction of the phaZ lesion into the phasin mutant background was used to construct a phaZ-phasin mutant strain. Analysis of the PHB biosynthesis capacity of this strain showed that the lack of PHB synthesis exhibited by S. meliloti phasin mutants is due to loss of PHB biosynthesis activity and not due to an inherent instability in the PHB granules themselves. A recent study suggested that some bacteria may possess an alternate pathway for acetate assimilation that would bypass the need for the glyoxylate cycle in organisms that do not possess the enzyme, isocitrate lyase. In these organisms, acetate is assimilated through the ethylmalonyl-CoA pathway, which has significant overlap with the anabolic half of the PHB cycle, including reliance on the PHB intermediate 3-hydroxybutyryl-CoA. The observation that phbB and phbC mutants of S. meliloti are unable to grow well on acetoacetate -- coupled with previously unexplained data that show a class of mutants (designated bhbA-D) are able to grow on acetate, but not on hydroxybutyrate or acetoacetate -- made it tempting to speculate that an ethylmalonyl-CoA-like pathway might be present in S. meliloti, and that this pathway might overlap with the PHB cycle at the point of 3-hydroxybutyryl-CoA. An in-frame mutation of phbA was constructed by cross-over PCR and allelic replacement. This mutant exhibited a complete abolition of growth on acetoacetate, suggesting that PhbA represents the only exit point for carbon from the PHB cycle and that an alternative ethylmalonyl-CoA-like pathway is not present in this organism. During symbiosis, rhizobial cells are dependent on the provision of carbon from the host plant in order to fuel cellular metabolism. This carbon is transported into the bacteroids via the dicarboxylate transport protein, DctA. Most rhizobia possess single copies of the transporter gene dctA and its corresponding two-component regulatory system dctBD. The completed genome sequence of B. japonicum suggests that it possesses seven copies of dctA. Complementation of Sinorhizobium meliloti dct mutants using the cosmid bank of B. japonicum USDA110 led to the identification a dctA locus and a dctBD operon. Interestingly, the B. japonicum dctABD system carried on the complementing cosmid was not able to complement the symbiotic deficiency of S. meliloti strains carrying individual mutations in either dctA, dctB, or dctD suggesting that the B. japonicum dctBD is unable to recognize either DctB/DctD or the DctB/DctD-independent regulatory elements in S. meliloti. All seven B. japonicum dctA ORFs were cloned and an analysis of their capacity to complement the free-living phenotype of a S. meliloti dctA mutant demonstrated that they all possess some capacity for dicarboxylate transport. Mutants of all seven B. japonicum dctA ORFs were constructed and an analysis of their free-living phenotypes suggested that significant functional redundancy exists in B. japonicum DctA function. Given the large number of potential dctA genes in the genome, coupled with an apparent lack of dctBD regulators, it is tempting to speculate that different DctA isoforms may be used during free-living and symbiotic growth and may be subject to different regulatory mechanisms than those of better-studied systems. A comprehensive analysis of desiccation tolerance and ion sensitivity in S. meliloti was conducted. The results of these analyses suggest that genetic elements on both pSymA and pSymB may play a significant role in enhancing cell survival under conditions of osmotic stress. The S. meliloti expR+ strains SmUW3 and SmUW6 were both shown to exhibit considerably higher desiccation tolerance than Rm1021, suggesting a role for enhanced exopolysaccharide production in facilitating survival under adverse conditions. Furthermore, scanning electron microscopy of inoculated seeds suggests that S. meliloti cells initiate biofilm formation upon application to the surface of seeds. This finding has implications for the analysis of OSS and the development of desiccation assays and may explain some of the variability that is characteristic of desiccation studies.

Most members of the Rhizobiaceae possess single copies of the poly-3-hydroxybutyrate biosynthesis genes, phbA, phbB and phbC. Analysis of the genome sequence of Bradyrhizobium japonicum reveals the presence of five homologues of the PHB synthase gene phbC as well as two homologues of the biosynthesis operon, phbAB. The presence of multiple, seemingly redundant homologues may suggest a functional importance. Each B. japonicum phbC gene was cloned and used to complement the pleiotropic phenotype of a Sinorhizobium meliloti phbC mutant; this mutant is unable to synthesize PHB, grow on certain PHB cycle intermediates and forms non-mucoid colonies on yeast mannitol medium. Two of the five putative B. japonicum phbC genes were found to complement the S. meliloti phbC mutant phenotype on D-3-hydroxybutyrate although none of them could fully complement the phenotype on acetoacetate. Both complementing genes were also able to restore PHB accumulation and formation of mucoid colonies on yeast mannitol agar to phbC mutants. In-frame deletions were constructed in three of the five phbC open reading frames in B. japonicum, as well as in both phbAB operons, by allelic replacement. One of the phbC mutants was unable to synthesize PHB under free-living conditions; one of the two phbAB operons was shown to be necessary and sufficient for PHB production under free-living conditions. These mutants also demonstrated an exopolysaccharide phenotype that was comparable to S meliloti PHB synthesis mutants. These strains were non-mucoid when grown under PHB-inducing conditions and, in contrast to wild-type B. japonicum, formed a compact pellet upon centrifugation. Interestingly, none of the mutants exhibited carbon-utilization phenotypes similar to those exhibited by S. meliloti PHB mutants. Wild-type B. japonicum accumulates PHB during symbiosis, and plants inoculated with the phbC mutants demonstrate a reproducible reduction in shoot dry mass. Analysis of bacteroid PHB accumulation in the mutant strains suggests that the phbAB operons of B. japonicum are differently regulated relative to growth under free-living conditions; mutants of the second phbAB operon demonstrated a significant reduction in PHB accumulation during symbiosis. These data suggest that the first phbAB operon is required for PHB synthesis only under free-living conditions, but is able to partially substitute for the second operon during symbiosis. Deletion of both phbAB operons completely abolished PHB synthesis in bacteroids. Analysis of the upstream regions of these genes suggest the existence of putative RpoN binding sites, perhaps indicating a potential mode of regulation and highlighting the metabolic complexity that is characteristic of the Rhizobiaceae. PHB metabolism in S. meliloti has been studied in considerable detail with two notable exceptions. No reports of the construction of either a β-ketothiolase (phbA) or a PHB depolymerase (phaZ ) mutant have ever been documented. The phaZ gene, encoding the first enzyme of the catabolic half of the PHB cycle in S. meliloti, was identified and a phaZ mutant strain was generated by insertion mutagenesis. The phaZ mutant demonstrates a Fix+ symbiotic phenotype and, unlike other PHB cycle mutants, does not demonstrate reduced rhizosphere competitiveness. Bacteroids of this strain were shown to accumulate PHB, demonstrating for the first time that S. meliloti is able to synthesize and accumulate PHB during symbiosis. Interestingly, there is no significant difference in shoot dry mass of plants inoculated with the phaZ mutant, suggesting that PHB accumulation does not occur at the expense of nitrogen fixation. The phaZ mutant strain was also used to demonstrate roles for PhaZ in the control of PHB accumulation and exopolysaccharide production. When grown on high-carbon media, this mutant demonstrates a mucoid phenotype characteristic of exopolysaccharide production. Subsequent analyses of a phoA::exoF fusion confirmed elevated transcription levels in the phaZ mutant background. In contrast, mutants of the PHB biosynthesis gene, phbC, have a characteristically dry phenotype and demonstrate reduced exoF transcriptional activity. The phaZ mutant also demonstrates a significant increase in PHB accumulation relative to the wild-type strain. Previous work on phasin mutants in S. meliloti demonstrated that they lack the ability to synthesize PHB. Transduction of the phaZ lesion into the phasin mutant background was used to construct a phaZ-phasin mutant strain. Analysis of the PHB biosynthesis capacity of this strain showed that the lack of PHB synthesis exhibited by S. meliloti phasin mutants is due to loss of PHB biosynthesis activity and not due to an inherent instability in the PHB granules themselves. A recent study suggested that some bacteria may possess an alternate pathway for acetate assimilation that would bypass the need for the glyoxylate cycle in organisms that do not possess the enzyme, isocitrate lyase. In these organisms, acetate is assimilated through the ethylmalonyl-CoA pathway, which has significant overlap with the anabolic half of the PHB cycle, including reliance on the PHB intermediate 3-hydroxybutyryl-CoA. The observation that phbB and phbC mutants of S. meliloti are unable to grow well on acetoacetate -- coupled with previously unexplained data that show a class of mutants (designated bhbA-D) are able to grow on acetate, but not on hydroxybutyrate or acetoacetate -- made it tempting to speculate that an ethylmalonyl-CoA-like pathway might be present in S. meliloti, and that this pathway might overlap with the PHB cycle at the point of 3-hydroxybutyryl-CoA. An in-frame mutation of phbA was constructed by cross-over PCR and allelic replacement. This mutant exhibited a complete abolition of growth on acetoacetate, suggesting that PhbA represents the only exit point for carbon from the PHB cycle and that an alternative ethylmalonyl-CoA-like pathway is not present in this organism. During symbiosis, rhizobial cells are dependent on the provision of carbon from the host plant in order to fuel cellular metabolism. This carbon is transported into the bacteroids via the dicarboxylate transport protein, DctA. Most rhizobia possess single copies of the transporter gene dctA and its corresponding two-component regulatory system dctBD. The completed genome sequence of B. japonicum suggests that it possesses seven copies of dctA. Complementation of Sinorhizobium meliloti dct mutants using the cosmid bank of B. japonicum USDA110 led to the identification a dctA locus and a dctBD operon. Interestingly, the B. japonicum dctABD system carried on the complementing cosmid was not able to complement the symbiotic deficiency of S. meliloti strains carrying individual mutations in either dctA, dctB, or dctD suggesting that the B. japonicum dctBD is unable to recognize either DctB/DctD or the DctB/DctD-independent regulatory elements in S. meliloti. All seven B. japonicum dctA ORFs were cloned and an analysis of their capacity to complement the free-living phenotype of a S. meliloti dctA mutant demonstrated that they all possess some capacity for dicarboxylate transport. Mutants of all seven B. japonicum dctA ORFs were constructed and an analysis of their free-living phenotypes suggested that significant functional redundancy exists in B. japonicum DctA function. Given the large number of potential dctA genes in the genome, coupled with an apparent lack of dctBD regulators, it is tempting to speculate that different DctA isoforms may be used during free-living and symbiotic growth and may be subject to different regulatory mechanisms than those of better-studied systems. A comprehensive analysis of desiccation tolerance and ion sensitivity in S. meliloti was conducted. The results of these analyses suggest that genetic elements on both pSymA and pSymB may play a significant role in enhancing cell survival under conditions of osmotic stress. The S. meliloti expR+ strains SmUW3 and SmUW6 were both shown to exhibit considerably higher desiccation tolerance than Rm1021, suggesting a role for enhanced exopolysaccharide production in facilitating survival under adverse conditions. Furthermore, scanning electron microscopy of inoculated seeds suggests that S. meliloti cells initiate biofilm formation upon application to the surface of seeds. This finding has implications for the analysis of OSS and the development of desiccation assays and may explain some of the variability that is characteristic of desiccation studies.

Thiomicrospira crunogena, a deep-sea hydrothermal vent chemolithoautotroph, uses the Calvin-Bensen-Bassham cycle to fix carbon. To meet its biosynthetic needs for oxaloacetate, oxoglutarate, and succinyl-coA, one would expect that this obligately autotrophic Gammaproteobacterium would use a ‘wishbone’ version of the citric acid cycle (CAC) to synthesize the intermediates necessary for biosynthesis, instead of the fully oxidative version to minimize carbon loss as carbon dioxide. However, upon examination of its complete genome sequence, it became apparent that this organism did not fulfill this expectation. Instead of a wishbone pathway, T. crunogena appears to run a fully oxidative CAC. The cycle is ‘locked’ in the oxidative direction by replacement of the reversible enzyme malate dehydrogenase with malate: quinone oxidoreductase, which is capable only of operation in the oxidative direction. Furthermore, oxoglutarate decarboxylation is catalyzed by oxoglutarate: acceptor oxidoreductase. The presence of both oxidoreductases was confirmed via assays on T. crunogena cell extracts. To determine whether this peculiar CAC was novel, complete genome sequences of ~340 Proteobacteria were examined via BLAST and COG searches in the Integrated Microbial Genome database. Genes catalyzing steps in the CAC were collected from each organism and vetted for paralogs that had adopted an alternative, ‘non-CAC’ function through genome context and cluster analysis. Alignments were made with the remaining sequences and were verified by comparing them to curated alignments at Pfam database and examination of active site residues. Phylogenetic trees were constructed from these alignments, and instances of horizontal gene transfer were determined by comparison to a 16S tree. These analyses verified that the CAC in T. crunogena is indeed unique, as it does not resemble any of the canonical cycles of the six classes of proteobacteria. Furthermore, three steps of the nine in its CAC appear to be catalyzed by enzymes encoded by genes that are likely to have been acquired via horizontal gene transfer. The gene encoding citrate synthase, and perhaps aconitase, are most closely affiliated with those present in the Cyanobacteria, while those encoding oxoglutarate: acceptor oxidoreductase cluster among the Firmicutes, and malate: quinone oxidoreductase clusters with the Epsilonproteobacteria.

No description available.

Carbon catabolite repression (CCR) is a mechanism by which micro-organisms preferentially utilize more easily metabolizable carbon sources in comparison to less easily metabolizable carbon sources. It prevents the organisms from unnecessary expenditure of energy and enables them to exploit the nutrients in appropriate manner. It represents a complex system of gene regulation. The main aim of this study was to study the intracellular localization of proteins involved in CCR including CreA, CreB, CreC and CreD in A. nidulans in repressing and derepressing conditions. The major regulatory protein involved in CCR in A. nidulans is CreA. It is a DNA-binding repressor, but very little is known about the molecular events that allow CreA function to result in appropriate regulation in response to carbon source. To determine the amount and localization of CreA in different carbon sources, strains were made over-expressing GFP and HA tagged CreA. Western analysis showed that high levels of full length CreA can be present in cells that show normal responses to carbon catabolite repression, whether they are grown in repressing or derepressing media. Hence the amount of CreA is similar in both the conditions and thus degradation of CreA is not a key step in carbon catabolite repression. Fluorescence microscopy studies have shown that CreA is in the nucleus under repressing and derepressing carbon conditions and this is not affected by the absence of CreB or CreD, the other important proteins in A. nidulans. Thus mere localization of CreA in nucleus is not sufficient to cause carbon catabolite repression and there is some modification process involved for CreA to act as a repressor protein in CCR. CreB is a deubiquitinating protein and CreC is a protein containing five WD 40 repeats, a putative nuclear localization signal (NLS) and a proline rich region and both the proteins are present in the cell in a complex. CreB was localized using strains that over-expresses GFP tagged CreB and fluorescence microscopy. CreB is present mainly in the cytoplasm in both repressing and derepressing conditions. Moreover, intracellular localization of CreB is unaffected by the presence or absence of CreD. However, the amount of CreB was higher in a creD+ background as compared to a creD34 mutant background, implying that the presence of CreD affects the amount of CreB in the cell. CreC was localized by using strain that over-expresses YFP tagged CreC and it is also present mainly in the cytoplasm. CreD contains arrestin domains and PY motifs and is highly similar to the Rod1p and Rog3p from S. cerevisiae. CreD is proposed to be involved in ubiquitination process in CCR in A. nidulans. Localization studies have shown that CreD is present throughout the cell in a punctate pattern with more in the cytoplasm than in the nucleus. CreB and CreD co-localize in some regions of the cell whereas in other regions either CreB or CreD is present. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1346526 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008