Orientador: Silvia Yuko Eguchi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-07-22T14:28:10Z (GMT). No. of bitstreams: 1 Maeda_AlfredoHitoshi_M.pdf: 3409883 bytes, checksum: a7a2529cd543e1ba8cb8f9b6d5fb05ce (MD5) Previous issue date: 1997 / Resumo: Bactérias acéticas do gênero Acetobacter foram identificadas em Usina de Açúcar e Álcool. Do total de 32 linhagens classificadas como Acetobacter sp, 11 (34,37 %) foram isoladas da peneira do "Cush-Cush", 3 (9,38 %) foram isoladas da parede do tanque de coleta do caldo de cana recém peneirado no "CushCush", 15 (46,87 %) foram isoladas do caldo de cana recém peneirado e 3 (9,38 %) foram isoladas da parede do Separador. As linhagens identificadas como bactérias acéticas foram testadas quanto a capacidade fermentativa em processo descontínuo. A taxa específica de produção de ácidos e a produtividade foram analisadas a partir das curvas de crescimento celular e de produção de ácidos. As linhagens Acetobacter sp CCT 2910, CCT 2023 e CCT 2924 apresentaram altas taxas de produção de ácidos, após 48 horas de fermentação, e as melhores taxas específicas de produção de ácidos e produtividades entre as cepas estudadas neste trabalho / Abstract: Acetic acid bacteria of the Acetobacter genus have been identified at the sugarcane mill, in ethanol plants. Out of a total of 32 strains, classified as Acetobactersp., 11 (34,37 %) have been isolated in the Cush-Cush sieve, 3 (9,38 %) have been isolated from the collect tank walls of the newly filtered sugar juice in the Cush-Cush, 15 (46,87 %) have been isolated from newly filtered sugar cane and 3 (9,38 %) have been isolated from the Separator. The strains identified as acetic acid bacteria have been tested as for their fermenting capacity in a discontinued processo The specific rate of acid production and productivity have been analysed based on the cell growth chart and acid production. The CCT 2910, CCT 2023 and CCT 2924 Acetobacter sp. strains have showed high acid production rates after 48 hours of fermenting and the best specific rates of acid production and productivity among of the strains studied in this paper / Mestrado / Mestre em Ciência de Alimentos

<i>Gluconobacter oxydans</i> is characterized by the ability to carry out rapid, single-step oxidations of many different hydroxyl-containing compounds. These oxidations are believed to be catalyzed by the membrane-bound NAD(P)-independent dehydrogenases. Experiments were designed to use <i>G. oxydans</i> ATCC strain 621 to determine the contribution of these dehydrogenases in whole-cell oxidations and to determine the range of substrates that can be oxidized by the membrane fraction of these cells when grown on a single substrate. My first hypothesis was that the membranes would accomplish these oxidations at the same rate as an equivalent number of whole cells. Oxidative activity data obtained from using both oxygen uptake and tetranitroblue tetrazolium assays, however, did not support this hypothesis. I attribute this to the probability that the membranes were damaged during isolation of the membrane fraction and, therefore, were unable to exhibit full oxidative potential. My second hypothesis was that the membranes from cells grown on one substrate would oxidize many other substrates. Potassium fenicyanide was used to assay the oxidative activity of the membrane fraction of cells grown on glycerol. Of 41 substrates tested all were significantly oxidized. I concluded from these data, therefore, that the enzyme(s) responsible for the oxidation of these substrates are synthesized constitutively. Unfortunately, one cannot draw any conclusions as to whether or not these enzymes are highly substrate-specific. I speculate that one or a few enzymes show a broad range of substrate specificity, as it would be inefficient for the cell to consecutively synthesize more than forty different substrate-specific enzymes for substrates it may never encounter. / Master of Science

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Wine quality is greatly influenced by the number of microorganisms, which occur throughout the winemaking process. Yeasts are responsible for the alcoholic fermentation, the lactic acid bacteria (LAB) are responsible for malolactic fermentation (MLF), while acetic acid bacteria (AAB) are responsible for converting ethanol to acetic acid. These microorganisms are present on the grapes and in the cellar and these consequently serve as gateways to the fermentation tanks where they will affect the wine quality. However, these microorganisms can be seen either as beneficial or as wine spoilage microorganisms, depending on the conditions that prevail throughout the winemaking process. It is thus very important to prevent any process that could lead to the lowering of the wine quality. In this regard, some of the factors that should always be evaluated include the quality of the grapes, winemaking techniques and quality control. One of the measures that have been implemented during winemaking to ensure the microbial stability is the use of chemical preservatives. Sulphur dioxide (502) has been, and is, used widely as primary preservative in winemaking. However, an ever-increasing consumer resistance against the use of chemical preservatives has developed as it poses possible health risks and decreases the sensorial quality of wine. An alternative approach to chemical preservation that has triggered numerous new investigations, is biological preservation or biopreservation. This is the use of the natural microbial flora and/or their antimicrobial products, such as bacteriocins, to inhibit or destroy the other sensitive microorganisms that are unwanted in the same environment. Evidence in the wine industry has shown that bacterial spoilage still is a very common problem in many wineries. This bacterial spoilage can lead to, amongst other, two main problems, which are of great concern to winemakers. This include high levels of volatile acidity, resulting in the wine having a vinegary off-flavour, and sluggish/stuck fermentation, which is the result of compounds such as acetic- and other fatty acids that causes inhibition of the yeast's growth. With acetic acid being the common link in both cases, it became evident that investigations should be performed on the main producer of acetic acid, namely AAB. As a result, AAB turned out to be one of the main spoilage microorganisms associated with winemaking. Most of the research on biopreservation in the food and beverage industry has been performed on the Gram-positive LAB. The fact that their spectrum of inhibition currently excludes most Gram-negative bacteria, specifically AAB, indicated that AAB should be screened in search of possible antimicrobial compounds that could be applied to control their cell numbers during winemaking. No evidence of antimicrobial action amongst AAB could be found in literature, therefore this work was considered novel. The main objectives of this study were to screen wine isolates of AAB for the production of antimicrobial compounds. This was followed by the isolation and preliminary characterisation of the antimicrobial substances produced. Various attempts to optimise the production of the antimicrobial compounds and isolation procedures, were also included. This study forms part of a larger research programme that has been initiated at the Institute for Wine Biotechnology at Stellenbosch University on the biopreservation in wine. Our results indicated that possible antimicrobial compounds of proteinaceous nature, produced by AAB isolated from wine, do exist. It was found that two different species of AAB, namely Acetobacter aeeti and Gluconobacter frateurii, produced antimicrobial compounds that inhibited other species of AAB. Preliminary results indicated that these compounds are heat sensitive and stable in a wide pH range. It was also shown that after the action of proteolytic enzymes, such as proteinase K and a-chemotrypsin, all inhibitory activity was lost. This study also revealed the existence of the species Gluconobacter frateurii, which have not yet been associated with the winemaking environment. This study made a valuable contribution to the limited amount of information and understanding of AAB, not only in the wine environment, but also elsewhere. The results and findings of this research would serve as platform for further projects. This might soon lead to the development of antimicrobial substances or tailored wine-yeasts with antimicrobial abilities, which can be applied during winemaking to assist the winemaker in combatting high cell numbers and subsequent spoilage by AAB. / AFRIKAANSE OPSOMMING: Wynkwaliteit word beïnvloed deur 'n verskeidenheid van mikroorganismes wat regdeur die wynrnaakproses teenwoordig is. Die giste is vir die alkoholiese fermentasie, die melksuurbakterieë (MSB) vir die appelmelksuurgisting, terwyl die asynsuurbakterieë (ASB) vir die omskakeling van etanol na asynsuur verantwoordelik is. AI hierdie mikroorganismes is teenwoordig op die druiwe en in die kelder, en dit dien gevolglik as 'n weg waardeur hulle in die fermentasietenke kan kom om sodoende die wynkwaliteit te beïnvloed. Hierdie mikroorganismes kan egter gesien word as óf voordelig óf as wynbederfmikroorganismes, afhangende van die heersende kondisies gedurende die wynrnaakproses. Dit is daarom baie belangrik om enige proses te voorkom wat tot 'n verlaging in wynkwaliteit kan lei. Wat laasgenoemde aanbetref, is daar sekere faktore wat altyd geëvalueer moet word, naamlik die druifkwaliteit, wynrnaaktegnieke en kwaliteitsbeheer. Een van die maatreëls wat geïmplementeer is om mikrobiologiese stabiliteit tydens die wynrnaakproses te handhaaf, is die gebruik van chemiese preserveermiddels. Swaweidioksied (S02) word algemeen gebruik as primêre preserveermiddel tydens wynrnaak. Daar is egter 'n toenemende verbruikersweerstand teen die gebruik van chemiese preserveermiddels, aangesien dit moontlike gesondheidsrisiko's kan inhou, asook tot 'n verlaging in sensoriese kwaliteit van die wyn kan lei. 'n Alternatiewe benadering vir chemiese preservering, wat reeds tot verskeie nuwe ondersoeke gelei het, is biologiese preservering of biopreservering. Dit is die gebruik van die natuurlike mikroflora en/of hulle antimikrobiese produkte, soos bv. bakteriosiene, om die sensitiewe mikroorganismes wat in dieselfe omgewing voorkom, se groei te inhibeer óf om hulle dood te maak. Aanduidings vanuit die wynbedryf dui daarop dat bakteriese bederf steeds 'n algemene probleem is wat in baie kelders ondervind word. Hierdie bakteriese bederf kan onder andere twee hoofprobleme veroorsaak, wat 'n groot bekommernis vir verskeie wynmakers is. Dié probleme sluit in hoë vlakke van vlugtige suurheid, wat gevolglik die wyn 'n asyn-afgeur gee, en slepende/gestaakte fermentasies, wat die gevolg is van komponente soos asynsuur en ander vetsure, wat die gis se groei inhibeer. Die feit dat asynsuur die gemeenskaplike faktor in beide gevalle was, het daarop gedui dat 'n ondersoek rakende die hoofproduseerder van asynsuur, naamlik ASB, benodig word. ASB word gevolglik as een van die hoofbederforganismes wat met die wynrnaakproses geassosieer word, beskou. Die meeste navorsing oor biopreservering in die voedsel -en drank bedryf is op die Gram-positiewe MSB gedoen. Die spektrum van inhibisie van die bakteriosiene van MSB sluit egter die meeste Gram-negatiewe bakterieë uit, veral ASB, en dit dui daarop dat ASB gesif moet word in 'n soektog na antimikrobiese substanse wat moontlik gebruik kan word om hul getalle tydens die wynrnaakproses te beheer. Geen bewyse kon tot dusver uit die literatuur gekry word met betrekking tot antimikrobiese aktiwiteit teen ASB nie, daarom word hierdie navorsing dus as nuut beskou. Hierdie studie se hoofdoelwittewas om die wyn-isolate van ASB vir die produksie van antimikrobiese peptiede te sif. Dit is gevolg deur die isolasie en voorlopige karakterisering van die geproduseerde antimikrobiese komponente. Daar is ook verskeie pogings aangewend om die produksie van die antimikrobiese substanse, asook die isolasieprosedures, te optimiseer. Hierdie studie vorm deel van 'n groter navorsingsprogram oor biopreservering van wyn wat deur die Instituut vir Wynbiotegnologie by die Universiteit van Stellenbosch geïnisieer is. Die resultate het daarop gedui dat antimikrobiese substanse van proteïenagtige aard, afkomstig vanaf wyn-isolate van ASB, wel bestaan. Daar is gevind dat twee veskillende spesies, naamlik Aeefobaefer aeefi en Glueonobaefer frafeurii, antimikrobiese peptiede produseer, wat ander spesies van ASB kan inhibeer. Voorlopige resultate het getoon dat hierdie substanse hitte-sensitief is en ook stabiel is oor 'n wye pH-reeks. Daar was ook aanduidings dat, ná die aksie van proteolitiese ensieme, soos bv. proteïnase K en a-chemotripsien, al die inhibitoriese aktiwiteit verlore gegaan het. Hierdie studie het ook die voorkoms van die spesies Glueonobaefer frafeurii aangedui, wat nog nie tot dusver met die wynrnaakomgewing geassosieer is nie. Hierdie studie maak 'n waardevolle bydrae tot die beperkte hoeveelheid inligting oor en begrip van ASB, nie net in die wynomgewing nie, maar ook in die algemeen in die natuur. Die bevindinge en resultate van hierdie navorsing sal as basis dien vir verdere projekte wat sal volg. Dit kan moontlik binnekort lei tot die ontwikkeling van antimikrobiese substanse, en ook pasgemaakte wyngiste met antimikrobiese vermoëns, wat tydens die wynrnaakproses gebruik kan word om sodoende die wynmaker in staat te stelom die hoë bakteriese getalle en die gevolglike bederf deur ASB, te beheer.

We have developed a new biofabrication process in which the precise control of bacterial motion is used to fabricate customizable networks of cellulose nanofibrils. This work describes how the motion of Acetobacter xylinum can be controlled by electric fields while the bacteria simultaneously produce nanocellulose, resulting in networks with aligned fibers. Since the electrolysis of water due to the application of electric fields produces the oxygen in the culture media far from the liquid-air boundary, aerobic cellulose production in 3D structures is readily achievable. Five separate sets of experiments were conducted to demonstrate the assembly of nanocellulose by Acetobacter xylinum in the presence of electric fields in micro and macro environments. This work demonstrates a new concept of bottom up material synthesis by control of a biological assembly process. / Master of Science

Membrane-bound NAD-independent alcohol dehydrogenase (NAD-independent ADH) from Acetobacter and Gluconobacter species has shown appreciable enantioselectivity on the oxidation of chiral primary and secondary alcohols. A NADIndependent alcohol dehydrogenase from mesophilic Acetobacter sp was isolated and characterized as a quinohaemprotein alcohol dehydrogenase QH-ADH. QHADH showed enantiopreference for the oxidation of (S)-enantiomer during the kinetic resolution of racemic secondary alcohols. Due to the growing interest for thermotolerant enzymes in the biocatalysis area, this study was performed to isolate acetic acid bacteria from vinegar factories in the northeast region of Brazil aiming to obtain a thermotolerant QH-ADH. Among the strains isolated, two showed characteristics of the Acetobacter genus (LBVE1 e LBVE4). Biomass production of the two strains were conduced on shaker flasks assays with three different ethanol concentrations as carbon source (10, 15 e 20 g/L) incubated in three different temperatures (30 35 e 37ºC). The highest biomass production for the two strains was obtained on the assays with 20 g/L ethanol, 30 ºC. However, both strains presented different growth conductions for the production of biomass highly active in NADindependent ADH. LBVE1 biomass produced on 10 g/L ethanol, 30ºC, showed the highest specific activity, 10.44 U/mg Proteín while the highest specific activity for LBVE4, 8.04 U/mg Proteín, was obtained on the biomass produced in assays with 15 g/L ethanol, 30 ºC. The results of the substrate specificity of the NAD-independent ADH present in the crude extract of both strains for selected primary and secondary alcohols. For primary alcohols, LBVE1 crude extract showed a decrease in the activity values with the rise of the carbon chain. Kinetic assays were performed to calculate apparent kinetics constants, using ethanol as substrate, for the NADindependent ADH present in the crude extract of both strains. LBVE1 crude extracts from cells grown in 10 and 20 g/L ethanol showed comparable values for Kmapp and VMax app. However, only extract from LBVE4 grown in 20 g/L ethanol had Kmapp value comparable to the one found for LBVE1 extracts analyzed. / Fundação de Amparo a Pesquisa do Estado de Alagoas / Álcool desidrogenase NAD-independente, enzima ligada a membrana, das espécies Acetobacter e Gluconobacter mostram apreciável enantioseletividade na oxidação de álcoois quirais primários e secundários. Em estudos anteriores, esta enzima foi isolada e caracterizada de espécies mesofílicas Acetobacter sp como uma quinohemoproteína álcool desidrogenase (QH-ADH). QH-ADH mostrou enantiopreferência pela oxidação do (S)-enantiômero durante a resolução cinética de álcoois secundários racêmicos. Devido ao crescente interesse por enzimas termotolerantes na área de biocatálise, este estudo foi conduzido com intuito de isolar bactérias termotolerantes do ácido acético de fábricas de vinagre na região nordeste do Brasil, almejando obter uma QH-ADH termotolerante. Dentre as espécies isoladas, duas mostraram características do gênero Acetobacter (LBVE1 e LBVE4). A produção de biomassa foi conduzida em agitador sob aeração de 200 rpm com três diferentes concentrações de etanol (10, 15 e 20 g/L) e incubadas em três temperaturas (30 35 e 37ºC). Os dois isolados apresentaram maior produção de biomassa nas condições quando cultivadas a 20 g/L etanol na 30ºC. Todavia, as condições para produção de uma biomassa ativa em álcool desidrogenase NADindependente variaram para as duas cepas. Cultivos de LBVE1 com 10 g/L de etanol, 30ºC, apresentou maior atividade específica cerca de 10,44 U/mgProteína. Enquanto que cultivos de LBVE4 mostrou atividade específica mais alta, 8,04 U/mgP nos cultivos a 15 g/L de etanol, 30ºC. As análises para investigar os perfis de especificidades para o substrato nos extratos brutos de LBVE1 e LBVE4, contendo ADH NAD-independente foram realizadas usando álcoois racêmicos com diferentes tamanhos de cadeia carbônica, dentre eles o rincoforol que é utilizado como feromônio. Também foram medidos os parâmetros cinéticos aparentes nos extratos brutos de ambas as cepas contendo atividade em ADH NAD-independente. Os extratos brutos células de LBVE1 cultivados 10 g/L e 20 g/L etanol apresentaram valores de Kmapp e VMax app comparáveis. Contudo, somente os extratos brutos de células de LBVE4 cultivados a 20 g/L etanol tiveram valores de Kmapp semelhantes aos encontrados para os extratos de células de LBVE1 estudados.

The endosymbiotic bacteria Wolbachia and the gut microbiome have independently been shown to affect several aspects of insect biology, including reproduction, development, lifespan, stem cell activity and resistance to human pathogens in insect vectors. This research shows that Wolbachia, which reside mainly in the fly germline, affect the microbial species present in the gut of a lab reared strain of Drosophila melanogaster. Fruit flies host two main genera of commensal bacteria – Acetobacter and Lactobacillus. Wolbachia-infected flies have significantly reduced titers of Acetobacter. Analysis of the microbiome of axenic flies fed with equal proportions of both bacteria shows that Wolbachia’s presence is a determinant in the microbiome composition throughout fly development. This effect of Wolbachia on the Drosophila microbiome is host genotype-dependent. To investigate the mechanism of microbiome modulation, the effect of Wolbachia on Imd and ROS pathways, the main regulators of immune response in the fly gut was measured. Wolbachia’s presence did not cause significant gene expression changes of the effector molecules in either pathway. It was also found that Wolbachia slightly reduce the relative length of the acidic region of the gut. However, this observation lacks the robustness necessary to provide a mechanism for the significantly reduced Acetobacter levels. Furthermore, microbiome modulation is not due to direct interaction between Wolbachia and the gut microbes,as confocal microscopy shows that Wolbachia is absent from the gut lumen. These results indicate that the mechanistic basis of the modulation of microbiome composition by Wolbachia is more complex than direct bacteria interaction or Wolbachia’s effect on fly immunity. The findings reported here highlight the importance of considering the gut microbiome composition and host genetic background during Wolbachia-induced phenotypic studies and microbial based-disease vector control strategies.

The acsD gene is a unique gene present in the cellulose biosynthesis operon in G. xylinus. With the use of homologous recombination, the acsD gene disruption mutation was created in the G. xylinus genome. Phenotypic characterization of the acsD gene mutant was investigated with the assistance of light and electron microscopy observations, carboxymethyl cellulose alterations, and lower temperature incubation. The microscopic analysis of the cellulose ribbons secreted from the acsD gene mutant shows that the polymerization and the crystallization components in mutant cells were functional. Observations of the mutant cells after incubation with carboxymethyl cellulose and temperature changes indicate that the arrangements of the pores on the cell surface have been altered. These arrangements led to decreased cellulose secretion capacity of the mutant cells. Successful complementation was achieved by using gene expression plasmids with green fluorescence protein tag in the acsD mutant background. Anti-GFP antibodies were used to determine the in vitro localization of the protein. Using subcellular fractionation and western blotting, the AcsD protein was found to be localized in the periplasm of the cells. Taking all these results together, a new model for bacterial cellulose biosynthesis has been suggested and discussed. / text

Memoria para optar al título de Ingeniero en Alimentos / La uva de mesa es una de las principales especies frutales en Chile, ya que es uno de los mayores exportadores a nivel mundial y líder en el hemisferio sur. La principal enfermedad que ha afectado en Chile a la uva ha sido la Pudrición Gris, pero una nueva patología de la vid denominada "Pudrición ácida" ha aparecido progresivamente en la zona centro-norte. La etiología de esta fitopatología indica que los responsables serían hongos, levaduras y bacterias acéticas, sin haber determinado los roles y mecanismos de acción de estos microorganismos. En un estudio realizado en el INTA (Instituto de Nutrición y Tecnologia de los Alimentos), se encontró presencia de bacterias acéticas en un 52% de las uvas enfermas y se obtuvieron recuentos bajos de hongos filamentosos fitopatógenos en uvas enfermas, sugiriendo revisar las teorías sobre un origen principalmente fungoso. Luego, se investigó acerca de la asociación de las principales especies de acetobacterias, obteniéndose un predominio de Acetobacter aceti, con un 71% de presencia en uvas enfermas. Hasta el momento no se dispone de tratamientos eficaces que eviten el desarrollo de la pudrición ácida. Por eso, es que en el presente estudio se busca un método alternativo, amigable con el medio ambiente denominado “control biológico”. Para ello se seleccionaron un grupo de 42 cepas provenientes de la microbiota natural de vides, potenciales biocontroles por tener actividad inhibitoria contra BGA (Bacillus subtilis) y Escherichia coli. De estas, hubo 14 que manifestaron cierta propiedad antágonica contra sobre las bacterias acéticas. No se encontró actividad inhibitoria en el sobrenadante libre de células de los biocontroles, lo que sugiere que el principal mecanismo de acción de antagonismo, no sería la producción de metabolitos antibióticos. En las cepas potenciales biocontroles, no se halló un nivel importante de resistencia a antibióticos (solamente resistencia a la ampicilina y la eritromicina en 3 cepas); encontrándose presencia de plásmidos en 4 cepas (ninguno relacionado con el desarrollo de un cuadro de multidrogo-resistencia); y siendo todos los biocontroles pertenecientes al género Bacillus con predominio de Bacillus circulans y licheniformis / The table grape is one of the main fruit species in Chile, since it is one of the greater exporters of this fruit at world-wide level and leader in the South hemisphere. The main disease that has affected in Chile the grape has been the Grey Mold, but a new pathology denominated “Sour rot” has progressively appeared in the center-north zone. The etiology of this pathology indicates that “Sour rot” would be caused by yeasts, molds and acetic bacterias, without to have determined the roles and mechanisms of action of these microorganisms. In a study carried out in the INTA, presence of acetic bacteria in a 52% of the sick grapes was found and low recounts of filamentous molds were obtained in sick grapes, suggesting revising the theories on a mainly fungous origin. Then, thet investigated about the association of the main species of acetobacterias, being obtained that dominated Acetobacter aceti, with a 71% of presence in sick grapes. Until now there aren´t completely effective treatments that controls the development of the sour rot. For that reason, the present study looks for an alternative method, friendly with environment denominated “biological control”. Therefore, was selected a group of 42 environmental strains native of grapevines, potentials biocontrols with inhibiting activity against BGA (Bacillus subtilis) and Escherichia coli. Of these, there were 14 that showed certain antagonistic activity against the acetic bacterias. It wasn´t detected inhibiting activity in the cell-free supernadant, which suggests the main mechanism of antagonism action; it would not be the production of antibiotic metabolites. In the 14 potential biocontrols strains was not found an important presence of resistance to antibiotics (only resistance to the ampiciline and the eritromicine in 3 strains); there was presence of plasmids in 4 strains (not related to the development of a multidrug resistance); and all biocontrols belonged to the genus Bacillus with predominance of Bacillus circulans and Bacillus licheniformis

Research in our lab has predicted hundreds of bacterial genes that influence nine different traits in the fruit fly, Drosophila melanogaster. As a practical alternative to creating site-directed mutants for each of the predicted genes, we created an arrayed transposon insertion library using a strain of Acetobacter fabarum DsW_054 isolated from fruit flies. Creation of the Acetobacter fabarum DsW_054 gene knock-out library was done through random transposon insertion, combinatorial mapping and Illumina sequencing. Successful mapping of transposon insertion was achieved for 6418 mutants with hits within 63% of annotated genes within Acetobacter fabarum DsW_054. Insertion sites were verified in 40 mutants through arbitrary PCR and sequencing. To test the utility of the library, genes were selected from MGWAS results on host colonization which show LPS pathway enrichment in the significant gene predicctions. Genes upstream of Lipid-A creation show significant differences in host colonization whereas downstream genes show no effect. In addition, genes were selected from MGWAS results on Drosophila starvation resistance which show Methionine/Cysteine synthesis, Cobalamin synthesis, and Biotin synthesis pathway enrichment. Under our experimental conditions we could not verify influence of these pathways on host starvation resistance. However, they do appear to influence host colonization abundance. This transposon insertion mutant library will be useful for ongoing research in our lab as well as any field studying Acetobacter species, such as other insect microbiome and fermentation research.

Thesis (PhD (Biochemistry))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: In the last decade a need arose to find a theoretical framework capable of gaining a quantitative understanding of ecosystems. Control analysis was proposed as a suitable candidate for the analysis of ecosystems with various theoretical applications being developed, i.e. trophic control analysis (TCA) and ecological control analysis (ECA). We set out to test the latter approach through experimental means by applying techniques akin to enzyme kinetics of biochemistry on a simple ecosystem between Saccharomyces cerevisiae and Gluconobacter oxydans. However, this exercise was far more complex than we originally expected due to the extra metabolic activities presented by both organisms. Nevertheless, we derived suitable kinetic equations to describe the metabolic behaviour of both organisms, with regards to the activities of interest to us, from pure culture experiments. We developed new techniques to determine ethanol and oxygen sensitivity of G. oxydans based on its obligately aerobic nature. These parameters were then used to build a simple kinetic model and a more complex model incorporating oxygen limited metabolism we observed at higher cell densities of G. oxydans. Our models could predict both situations satisfactorily for pure cultures and especially the more complex model could describe the lack of linearity observed between metabolic activity and cell density at higher cell densities of G. oxydans. Mixed populations of S. cerevisiae and G. oxydans reached quasi-steady states in terms of ethanol concentration and acetate flux, which was a positive indication for the application of control analysis on the ecosystem. However, the theoretical models based on parameters derived from pure culture experiments did not predict mixed culture steady states accurately. Careful analysis showed that these parameters were mostly under-estimated for G. oxydans and overestimated for S. cerevisiae. Hence, we calculated the kinetic parameters for mixed population assays directly from the experimental data obtained from mixed cultures. We could calculate the control coefficients directly from the experimental data of mixed population studies and compare it with those from theoretical models based on 3 different parameter sets. Our analysis showed that the yeast had all the control over the acetate flux while control over the steady-state ethanol was shared. The strength of our approach lies in designing our experiments with a control analysis approach in mind, but we have also shown that even for simple ecosystems this approach is non-trivial. Despite the various experimental challenges, this approach was very rewarding due to the extra information obtained especially regarding control structure with regards to the steady-state ethanol concentration. / AFRIKAANSE OPSOMMING: In die afgelope dekade het daar ’n behoefte ontstaan na ‘n teoretiese raamwerk om tot ‘n kwantitatiewe begrip van ekosisteme te kom. As kandidaat vir so tipe raamwerk is kontrole analise voorgestel gepaardgaande met die ontwikkeling van verskeie teoretiese toepassings, i.e. trofiese kontrole analise en ekologiese kontrole analise. In hierdie tesis het ons laasgenoemde aanslag eksperimenteel ondersoek op ‘n eenvoudige ekosisteem, tussen Saccharomyces cerevisiae en Gluconobacter oxydans, deur gebruik te maak van tegnieke vanuit ensiemkinetika van biochemie. Hierdie strategie was egter baie meer kompleks as wat oorspronklik verwag is as gevolg van verdere metabolise aktiwiteite aanwesig in beide organismes. Ons het egter steeds daarin geslaag om kinetiese vergelykings af te lei, vanuit suiwer kulture, wat die metaboliese gedrag van beide organismes beskryf vir die aktiwiteite van belang vir ons studie. Ons het nuwe tegnieke, gebaseer op die aerobiese natuur van G. oxydans, ontwikkel om die sensitiwiteit van G. oxydans vir etanol en suurstof te bepaal. Hierdie parameters is gebruik om eers ’n eenvoudige model en toe ‘n meer gevorderde model, wat die suurstof-beperkte metabolisme van G. oxydans by hoër biomassa te beskryf, op te stel. Beide modelle was baie effektief in die voorspelling van die situasies waarvoor hulle ontwikkel is vir die suiwer kulture waar veral die meer gevorderde model die gebrek aan ‘n linieêre verband tussen die metabolisme van G. oxydans en biomassa by hoër biomassa kon beskryf. ’n Bemoedigende aanduiding dat kontrole analise toegepas kon word op die ekosisteem was dat mengkulture van S. cerevisiae en G. oxydans het quasi-bestendige toestande bereik het in terme van etanol konsentrasies en asetaat-fluksie. Die teoretiese modelle gebaseer op die parameters afgelei vanaf suiwer kulture kon egter nie die bestendige toestande in mengkulture akkuraat voorspel nie. Nadere ondersoek het aangedui dat die parameters meesal onderskat is vir G. oxydans en oorskat is vir S. cerevisiae. Gevolglik het ons die kinetiese parameters vir mengkulture direk van eksperimentele data van die mengkulture bereken. Verder kon ons die kontrole koeffisiente ook direk vanaf die eksperimentele data van mengkulture bereken en vergelyk met dié bereken vanuit die teoretiese modelle gebaseer op drie verskillende paremeter-stelle. Ons analise het gewys dat die gis alle beheer op die asetaat-fluksie uitoefen en dat die beheer oor die etanol-konsnetrasie gedeel is tussen die twee organismes. Die krag van ons aanslag lê daarin dat die eksperimente ontwerp is met ‘n kontrole analise in gedagte, maar ons het ook bewys dat hierdie aanslag selfs vir eenvoudige ekosisteme nie triviaal is nie. Ten spyte van die eksperimentele uitdagings, was die aanslag baie waardevol as gevolg van die ekstra inligting verkry met spesifieke klem op die kontrole-struktuur met betrekking tot die etanol konsentrasie by bestendige toestand.