There are six known secretion systems in Gram negative bacteria, referred to as Type 1 to Type 6 respectively, which are dedicated to moving substrate across the outer membrane. Secretion systems are broadly separated into those that move their substrate across the cell envelope in a single translocation event (one-step systems), and those that are dependent on the Sec or Tat machineries for export to the periplasm (two-step systems). Serratia marcescens is an important opportunistic human pathogen and has gathered a lot of interest due to its repertoire of secreted proteins. These include the haem-scavenging protein HasA, which is secreted by a Type 1 secretion system, and the cytotoxic haemolysin ShlA, which is secreted as part of a two-partner Type 5 secretion system. Serratia marcescens also encodes a Type 6 secretion system, which is known to translocate at least six effector molecules directly into other bacterial target cells. Serratia marcescens is a model organism in terms of its ability to degrade the quite intractable polymer chitin, for which it produces three chitinase enzymes ChiA, ChiB, ChiC and a chitin-binding protein Cbp21, which hydrolyse the ß-1,4 link in the chitin chain and promote binding of chitinase to the chitin substrate respectively. These chitinolytic enzymes are utilised by S. marcescens for both basic physiology and also in pathogenesis. In this work, genetic, biochemical and proteomic approaches identified, for the first time, genes that are essential for the secretion of all three chitinases as well as Cbp21. A genetic screen identified genes encoding a holin-like membrane protein (ChiW) and a putative L-alanyl-D-glutamate endopeptidase (ChiX). Subsequent quantitative proteomics experiments and biochemical analyses established that ChiW and ChiX were required for secretion of the entire chitinolytic machinery. Chitinase secretion was observed to be blocked at a late stage in the mutant strains as normally secreted enzymes were found to accumulate in the periplasm, thus implicating ChiW and ChiX in a novel outer membrane protein translocation process. It is proposed that the bacterial genome-encoded holin-like protein and endopeptidase identified represent a putative secretion system utilised by Gram-negative bacteria. In addition to this, genes encoding the chitinolytic machinery and the putative secretion apparatus were shown to be bimodally regulated and co-ordinately expressed.

C. albicans is a polymorphic fungal pathogen which has the ability to shift from yeast to hyphae. C. albicans cell wall is composed of glucan, chitin, mannoprotein and mannan. It is not possible, using standard extraction methods, to isolate mannan from C. albicans hyphae. To isolate hyphal mannan, we developed a simplified alkali extraction method. Using this method it was determined that hyphal mannan has a much lower molecular weight, a smaller polymer distribution and altered conformation structure when compared to yeast mannan. The hyphal mannan was found to contain little to no acid-labile portion with only α-Man-PO4 groups and no long chains of β-1, 2-linked mannosyl repeat units, when compared to the yeast mannan. It was concluded that the C. albicans hyphal mannan is substantially different from the mannan found in the yeast form. This is an entirely new observation that extends the existing knowledge about the structural biology of C. albicans hyphae and may provide insights into the role of hyphae in pathogenesis.

No description available.

Abstract The surface of multicellular organisms is covered with epithelial cells that provide a barrier to the external environment. As part of this barrier function, most epithelia produce apical extracellular matrices (aECMs). The generation of such chemical and physical barriers requires specialized deposition of macromolecules and is likely to involve a spatial and temporal coordination of biochemical activities at the apical surface. A challenging task is thus to characterize key proteins that underlie apical cell surface organization and correct aECM assembly. The Drosophila trachea provides an excellent system to study aECM formation, as they produce an ordered aECM, called the cuticle. The tracheal cuticle is unique by its presence of cuticular ridges, called taenidial folds, which prevent collapse of tracheal tubes while allowing them to expand and contract along their length. A gene called filzig encodes a transmembrane serine protease and is required for taenidial organization. The aim of this research was to further understand Filzig function through characterization of filzig mutants and Filzig protein expression. The results showed that Filzig is expressed in cuticle-producing epithelia as cuticle deposition begins. Moreover, Flz localized to the apical epithelial surface, as well as to the aECM. The apical Flz localization does not reflect the pattern of cuticle ridges, indicating that Flz-localization is not a determinant for taneidial patterning. Instead, Flz might act on extracellular targets that localize to the future taneidial folds. Alternatively, Filzig is involved in a cascade of self-organizing activity of cuticular components to form the regular taenidial folds.

Three experiments were conducted to evaluate the effects of dietary chitin and chitosan on growth, immune responses and resistance of grass prawn Penaeus monodon against white spot syndrome virus (WSSV) and Vibrio infections. In the first experiment, two levels (0.5¡B1 g/100g diet) of chitin and three levels (0.5¡B1¡B5 g/100g diet) of chitosan were evaluated. The results show that weight gain of the shrimp fed on diet containing no chitosan or the lowest level of chitosan (0.5 %) was higher than other groups. In the second experiment, four levels of chitosan (0¡B0.5¡B1¡B5 g/100g diet) were tested. Weight gains of the control (0 %) and 0.5 % chitosan groups were significantly (P<0.05) higher than the 0.1 and 1 % chitosan groups. Shrimp survival rate was not influenced by chitosan inclusion. The test shrimp of the first experiment were evaluated for their immune responses after dietary exposures. The results show that phenoloxidase activity and superoxide dismutase were not significantly different (P>0.05) among treatments. The production of superoxide anion in the 0.5 % chitin group was significantly (P<0.05) lower than the other groups at day 3 and 12. The last experiment evaluated the effectiveness of dietary chitosan against infection of WSSV and Vibrio damsela. Shrimp were fed for 20 days on test diets containing four levels of chitosan (0¡B0.5¡B1¡B5 g/100g diet) and then challenged by injection of WSSV or Vibrio solution. In the WSSV challenge, except at day 7, shrimp survivals were not different among treatments. At day 7, however, the survival rates of the shrimp fed the diet containing 0.1 or 1 % chitosan were significantly (P<0.05) higher than those of the other groups. When challenged with Vibrio damsela, there was no difference in shrimp survival among dietary treatments. The present study shows that dietary chitin and chitosan do not significantly enhance immune responses and disease resistance of juvenile P. monodon. Dietary incorporation of chitin or chitosan negatively affects shrimp growth.

Developing renewable materials to reduce the dependence on fossil fuel as a feedstock for a wide range of applications is becoming increasingly acknowledged as important in society. Chitin, the second most abundant biopolymer in nature, is an ideal candidate for diverse applications because of its remarkable properties, such as abundance, renewability, biodegradability, biocompatibility, antibacterial activity, chemical functionality, and high stiffness and strength. Despite these inherent advantages, chitin is currently still underutilized mainly due to its strong molecular interactions, which make it insoluble in common solvents. Currently, its major applications are limited to biomedical engineering, such as tissue engineering, wound dressing and sutures. This thesis aims to explore and enable the potential utilization of chitin in other fields where it may serve as a renewable functional advanced material. Here, a number of novel chitin-based materials were developed successfully without employing chitin dissolution. These include chitin nanofibers (CNFs), porous chitin with tunable structures, chitin-reinforced polymer composites and chitin-stabilized aqueous foams. Moreover, the properties of these materials including interfacial, optical, thermal, and mechanical characteristics were determined, and their potential utilizations were demonstrated. Briefly, in chapter 2, CNFs with diameters of ~20 nm were successfully extracted from crab α-chitin by a high pressure homogenization process. The produced CNFs were dispersed well in water without forming strong network structures due to their electrostatic repulsions. The obtained CNF film has a high residue amount (40%) when heated up to 1000 ˚C. Meanwhile, it exhibited high optical transparency as well as great gas barrier properties. In chapter 3, on the basis of the obtained CNFs in chapter 2, versatile porous structures including oriented sheets and three-dimensional aperiodic nanofiber networks were achieved by using a freeze drying technique. Since the formation of nanofibrous structures cannot be predicted by the widely-used particle encapsulation model, a modified structure formation mechanism was proposed. In chapter 4, the structure-property relationships of the CNF/poly(ethylene oxide)(PEO) nanocomposites were established. We demonstrated that the CNFs formed network structures in PEO matrix and had hydrogen bonding interaction with PEO. The CNFs can greatly enhance the mechanical properties of PEO, such as elastic modulus and tensile strength. In chapter 5, the aqueous foams stabilized by high-aspect-ratio CNFs were developed. The created foams exhibited strong hindrance on film drainage, coalescence and disproportionation. The fibrillated CNFs alone were not able to stabilize air bubbles, but the addition of small amounts of valeric acids in CNF dispersion can make chitin foamable. The results clearly showed that valeric acid modified CNFs reduced the surface tension of aqueous dispersion and were attached at the air-water interface. Overall, this research has provided many new insights for the fabrication, characterization, and utilization of chitin, and has built a solid foundation for further exploiting chitin for diverse applications.

Chitin belongs to the most abundant biopolymers on earth where it has an important role as a structural element in crustaceans, insects, fungi and some phytoplankton. Missing evidence for long-term accumulation of chitin in nature implies fast turnover and as chitin is composed of aminosugar subunits it holds central roles in both carbon and nitrogen cycles. The aim of this thesis was to contribute to a better understanding of organic matter cycling by learning more about the diversity, function and ecology of bacteria that degrade chitin. A metagenome-enabled study of the spatial distribution of chitinolytic bacteria in aquatic ecosystems identified salinity as the major environmental factor for shaping their community composition. To address the role of alternative environmental variables controlling chitinolytic communities, a temporally resolved study was completed in a dimictic freshwater lake. Pronounced seasonal change in the indigenous chitinolytic community was observed and parallel measured environmental parameters pointed to the availability and crystalline form of chitin as significant controlling factors.  The different ecological niches occupied by microbes that utilize chitin for growth were studied in an experimental study. Single-cell quantification of chitinolytic cells and cells incorporating chitin hydrolysis products suggested that commensal use of chitin hydrolysis products without simultaneous chitinase activity could be an important ecological strategy in freshwater bacterioplankton communities. Members of the ubiquitous and often quantitatively dominant group of freshwater Actinobacteria Ac1 were identified as particularly active in this “cheater” lifestyle. Further experiments based on artificially created gradients in bacterial diversity demonstrated the importance of specific bacterial populations and community composition rather than overall community richness in controlling more specific functions such as chitin and cellulose degradation. To conclude, results of this thesis provide insight into the biogeography, niche-separation and species interactions of the functional community of chitin degraders and the influence of general bacterial diversity to the respective system functioning. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 700

This thesis examined the economics of producing high value bio-ingredients such as chitin and carotenoprotein from shrimp processing waste in Newfoundland. The shrimp waste in the province was estimated to be at least 37000 tons annually. A survey of shrimp processing plants in the province revealed that the waste generated was relatively pure with little or no foreign material. The economic engineering approach was employed to estimate the production cost of chitin and carotenoprotein at the laboratory and pilot scale levels. At the laboratory scale where 480 kg/year of raw material (shrimp waste) was processed, the cost of chitin and carotenoprotein was found to be $159/kg and $315/kg, respectively. At the pilot scale level, the cost of chitin and carotenoprotem was estimated to be $125/kg and $244/kg, respectively based on volume of 4800 kg/year. Sensitivity analysis was carried out to establish the cost variations due to changes in the quantity of starting raw material, labor cost and cost of laboratory supplies (chemicals and enzymes). The cost of chitin and caroteinoprotein showed a decreasing trend with increasing scale of production. An expert opinion survey was conducted with a selected panel of 9 experts from the shrimp processing industry, chitin related industry, and the academic/research community to determine the potential market of the high-grade chitin/chitosan in Canada. The results showed that the health and nutraceutical industry is the most promising niche for high-grade chit in/chitosan. The survey also indicated that potential market would be high in Ontario and Quebec due to the presence of large health and nutraceutical companies in the big metropolitan areas of these regions.

Recent preliminary data have suggested that microbe-to-plant signals, and plant internal signals elicited by microbial signals, affect aspects of plant physiology, development and growth. The reported research investigated the responses of plants to signal compounds of microbial and plant origin, such as lipo-chitooligosaccharides (LCOs - signal molecules in rhizobia-legume associations), chitin and chitosan (present in fungal cell walls), and phenolic compounds (salicylic acid, acetylsalicylic acid and gentisic acid - internal signals in plants, often affected by signals from microbes). Phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) are key enzymes of the phenylpropanoid pathway. Oligomers of chitin and chitosan increased the activities of both PAL and TAL in soybean leaves. The degree of increase was dependent on oligomer chain length and time after treatment. LCO [Nod Bj V (C18:1 , MeFuc)] was isolated from Bradyrhizobium japonicum strain 532C. When Arabidopsis thaliana plants were grown for two weeks on agar containing this LCO (10-8M) or chitin pentamer (10-4 M), they had greater root length, root diameter, root surface area and number of root tips than control plants. Chitosan (tetramer and pentamer) did not have this effect. Chitin and chitosan were also tested for effects on corn and soybean photosynthetic rates and growth. High molecular weight chitosan generally reduced photosynthetic rates, but did not reduce the growth of corn or soybean. However, foliar application of 10-6 M LCO to corn leaves increased photosynthetic rates (up to 36%). Foliar application of lumichrome (10-5 and 10-6 M), a breakdown product of riboflavin produced by some rhizosphere bacteria, to corn (C4 plant) and soybean (C3 plant) increased photosynthetic rates (up to 6%). Foliar application of lumichrome (10-5 M) increased soybean leaf area and shoot dry weight. Foliar application of SA, acetyl salicylic acid (ASA) and gentisic acid (GT

Thesis (M.S.)--University of Delaware, 2008. / Principal faculty advisor: Jeffry J. Fuhrmann, Dept. of Plant & Soil Science. Includes bibliographical references.