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In this project, Cytophaga hutchinsonii, an aerobic gliding bacterium with cellulose-degrading ability, was studied, since its cellulase system was unknown and might be very different from those of other cellulose-degrading species. Only ß-1,4- endoglucanases and non-specific ß-glucosidases were found in the C. hutchinsonii genome sequence, whereas specific exoglucanases were apparently absent. Almost all putative cellulases were composed of catalytic domains only, without carbohydrate-binding modules. Samples from C. hutchinsonii cultures were analyzed by using TLC and colorimetric assays. Glucose was detected in the cellobiose grown culture, but not in cellulose-grown cultures, suggesting that cellobiose is hydrolyzed extracellularly rather than being directly assimilated, and that cellulose may not be degraded via cellobiose. Also, cellobiose-based cultures caused greater acidification of the medium than glucose or cellulose grown cultures. Nine putative cellulases were expressed in four bacterial strains. In some cases, expression was toxic to host cells. The crude lysates were tested for endoglucanase, specific exoglucanase or nonspecific ß-glucosidase activity. CHU_1280 and CHU_1842 showed apparent endoglucanase activity when expressed in Citrobacter freundii. Four putative GH family 3 ß-glucosidases with similar conserved domains were expressed in Escherichia coli JM109 and E. coli BL21(DE3)pLysS. One of these, CHU_2268, was found to possess MUC-degrading ability. This suggests that CHU_2268 may be the 'missing' exoglucanase in C. hutchinsonii. Another two ß-glucosidases, CHU_2273 and CHU_3784, possessed only MUG-degrading activity.

Three strains of cyanobacteria (Anabaena PCC7120, A. variabilis and Nostoc commune), all belonging to the family Nostocaceae, were found to be capable of modulating the production and chemical composition of extracellular polysaccharides (EPS) in response to carbon and nitrogen availability as well as pH. While the carbohydrate compositions of the glycans produced by the different organisms were indicative of their recent evolutionary divergence, there were measurable differences that were dependent upon growth conditions. The EPS resulting from biofilm growth conditions was reduced in glucuronic acid levels in both Anabaena variabilis ATCC 29413 and Anabaena PCC 7120. Under planktonic conditions, the glycan from A. variabilis contained glucuronic acid when grown in nitrate-free BG-11₀ medium whereas A. PCC 7120 produced similar levels in standard BG-11 medium. This suggests that phylogeneticallyrelated cyanobacteria respond very differently to changes in their local environment. The pH of BG-11 cultures increased to 9-10 for all three strains of cyanobacteria. The increase resulted in an increase in the amount of dissolved inorganic carbon available in the medium, creating an imbalance in the carbon-nitrogen ratio, with the complete consumption of 17.65 mmol L⁻¹ nitrates raising the pH to near 10 in BG-11 medium. While increased carbon availability has been shown to induce capsulated morphologies in strains of cyanobacteria, only Nostoc commune DRH-1 exhibited this behavior, and only when grown in BG-11 medium. Carbon and nitrogen availability as well as pH modulate the monosaccharide composition of the glycan generated by cyanobacteria investigated. The different characteristics of the glycans produced can affect the survivability of the organisms and the community structure of cyanobacterial biofilms and microbial mats found in nature. As cyanobacteria are ubiquitous organism both now and in the past, they play a pivotal role in the biological and geological processes of the Earth, controlling the availability and cycling of carbon and nitrogen both actively and passively. / Master of Science in Life Sciences

In their natural environments microorganisms exist predominantly in aggregates and biofilms. The ability of bacteria to form aggregates is associated with the biosynthesis of polymers such as polysaccharides. In this study the physical mechanisms underlying bacterial aggregation by extracellular polysaccharides are investigated by utilising the bacterium Sinorhizobium meliloti. S. meliloti biosynthesises an extracellular polysaccharide called succinoglycan, which is well characterised in terms of its structure and biosynthesis. A range of previously constructed succinoglycan biosynthesis mutants were screened for altered aggregation. An S. meliloti exoS mutant (a gain of function mutation that results in a constitutively active two component regulator called ExoS) overproduces succinoglycan and has enhanced aggregation compared to the parent strain, Rm1021. The aggregates settle to the bottom of the culture vessel resulting in loss of turbidity of the cultures and phase separation. Microscopic observation showed that succinoglycan did not appear to be attached to the aggregates, which formed ordered structures of laterally aligned cells. By addition of purified succinoglycan it was found that the critical concentration of polymer required to induce aggregation and phase separation of the cultures decreased with increasing cell concentration. These observations suggest that aggregation of S. meliloti cultures in the presence of succinoglycan is driven by macromolecular crowding, otherwise known as depletion attraction. Depletion attraction can drive the ordered arrangement and aggregation of colloidal particles in the presence of polymers. Aggregation of the particles increases the volume available to the polymers, maximising their entropy and the entropy of the system. Addition of succinoglycan to stationary phase Escherichia coli cultures and polystyrene colloids also resulted in aggregation consistent with depletion attraction. Furthermore alternative polymers such as the bacterial extracellular polysaccharide xanthan produced by Xanthomonas campestris can result in aggregation of bacteria by depletion attraction. Depletion attraction may therefore be a ubiquitous force driving aggregation of crowded dispersions of bacteria and polymers. The second part of the thesis focuses on how depletion driven aggregation can lead to surface-associated biofilm formation. Imaging of the sediment formed by the exoS mutant showed that the structure formed at the base of the culture vessel leads to development of an ordered structure composed of interlinked aggregates. The role of succinoglycan in surface attachment is complex and varies with culture conditions. Depletion attraction may facilitate interaction with a surface but alternative factors may then play a role in anchoring the cells to the surface. Under certain conditions the cells may produce factors which allow binding of the cells to a surface independently of succinoglycan. This study has demonstrated for the first time that an extracellular polysaccharide produced by bacteria can result in aggregation via depletion attraction which may be an under explored mechanism by which aggregation of bacteria can occur.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23952号 / 農博第2501号 / 新制||農||1091(附属図書館) / 学位論文||R4||N5387(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 小川 順, 教授 阪井 康能 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM