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<p>Bacteria and fungi dominate the decomposition of aquatic plants, a major process in the carbon and nutrient cycling in many aquatic systems. Although phylogenetically distant, bacteria and fungi often live in close proximity with each other. Since these microorganisms also have similar ecological functions, interactions have developed between them. This thesis explores the nature of such interactions, and the potential effects on key components of the decomposition process. The thesis includes a critical assessment of the ergosterol method for determination of fungal biomass, a survey of the environmental factors determining the distribution and taxa numbers of litter-decomposing bacteria and fungi in lakes, and a number of experiments on the interactions between bacteria and fungi. In all the experiments performed, fungi responded to bacterial presence through antagonism, although different fungal strains, bacterial communities and substrates were used. The antagonism seemed to be caused by interference competition for substrate. The fungal effect on bacteria was less consistent. Bacterial growth was suppressed, unaffected, or even enhanced by the presence of fungi. Fungi contributed more to extracellular enzyme production than bacteria, and bacteria were probably able to assimilate intermediate decomposition products formed through the activity of extracellular enzymes of fungal origin. Thus, the effect on bacteria from interacting with fungi was determined by the balance between competition and benefit from excreted enzymes. Bacteria and fungi also used different size fractions of the organic matter, according to their different enzymatic capacities. Hence, bacteria appeared to assimilate low-molecular-weight compounds, while high-molecular-weight compounds were utilized primarily by fungi. </p><p>In brief, the ecological interactions influenced the growth and hence also the biomass development of bacteria and fungi, which affected enzyme activity as well as utilization of dissolved organic matter. Therefore, I suggest that interactions between bacteria and fungi influence degradation of plant litter in aquatic systems.</p>

Bacteria and fungi dominate the decomposition of aquatic plants, a major process in the carbon and nutrient cycling in many aquatic systems. Although phylogenetically distant, bacteria and fungi often live in close proximity with each other. Since these microorganisms also have similar ecological functions, interactions have developed between them. This thesis explores the nature of such interactions, and the potential effects on key components of the decomposition process. The thesis includes a critical assessment of the ergosterol method for determination of fungal biomass, a survey of the environmental factors determining the distribution and taxa numbers of litter-decomposing bacteria and fungi in lakes, and a number of experiments on the interactions between bacteria and fungi. In all the experiments performed, fungi responded to bacterial presence through antagonism, although different fungal strains, bacterial communities and substrates were used. The antagonism seemed to be caused by interference competition for substrate. The fungal effect on bacteria was less consistent. Bacterial growth was suppressed, unaffected, or even enhanced by the presence of fungi. Fungi contributed more to extracellular enzyme production than bacteria, and bacteria were probably able to assimilate intermediate decomposition products formed through the activity of extracellular enzymes of fungal origin. Thus, the effect on bacteria from interacting with fungi was determined by the balance between competition and benefit from excreted enzymes. Bacteria and fungi also used different size fractions of the organic matter, according to their different enzymatic capacities. Hence, bacteria appeared to assimilate low-molecular-weight compounds, while high-molecular-weight compounds were utilized primarily by fungi. In brief, the ecological interactions influenced the growth and hence also the biomass development of bacteria and fungi, which affected enzyme activity as well as utilization of dissolved organic matter. Therefore, I suggest that interactions between bacteria and fungi influence degradation of plant litter in aquatic systems.

"This thesis is based on the following articles, referred to in the text by the Roman numerals given below. In addition some unpublished results are presented. I. Caiyan Wu ... [et al] Improvement of the secretion of extracellular proteins and isolation and characterization of the amylase I (amyI) gene from Ophiostoma floccosum [pub. in ] Gene 384: 96-103 -- II. Caiyan Wu ... [et al.] Activity-based identification of secreted serine proteases of the filamentous fungus Ophiostoma. Accepted by Biotechnology letters DOI 10.1007/s10529-007-9333-6 -- III. Caiyan Wu ...[et al.] Expression of a thermostable bacterial xylanase in the filamentous fungus Ophiostoma floccosum. Submitted to Letters in applied microbiology in July 2007." - leaf 9. / Thesis (PhD)--Macquarie University, Division of Environmental and Life Sciences, Dept. of Chemistry & Biomolecular Sciences, 2007. / Bibliography: leaves 100-123. / Introduction -- Materials and methods -- Results and discussion -- Conclusion and future aspects -- References -- Publications I, II and III. / Ophiostoma spp. belong to the Ophiostomataceae family, a large group of ascomycetes, which are the most frequent blue stain fungi isolated from stained wood. Most Ophiostoma species do not compromise the strength properties of wood, but do reduce the aesthetic quality of timber and therefore decrease the economic value of lumber. Some albino variants of O. floccosum and O. piliferum have been used as biological control agents to prevent blue staining. This successful whole organism approach plus the added capability of extracellular protein secretion makes Ophiostoma spp. attractive for industrial application. In addition, Ophiostoma produces only a small range of abundantly secreted proteins in liquid culture, which can facilitate downstream purification of any recombinant gene product introduced into the system. Genes encoding efficiently secreted proteins provide a potential souce for strong promoters for high-level gene expression. These characteristics provide an excellent starting point for the development of a novel expression system. / In this study, UV-mutagenesis was applied to improve protein secretion in Ophiostoma floccosum. Amylase activity was used as an indicator for enhanced protein secretion after repeated rounds of mutagenic treatment. Several mutants of O. floccosum derived by UV mutagenesis were isolated and the total amount of secreted protein was increased by 4 to 6 times. The amylase activity in the culture supernatant of the best mutant (MQ.5.1) was increased by more than 240-fold compared to the initial parental strain. At the same time, the amount of total secreted protein was about six times greater to that of the parental strain. Proteinase profiles in the culture supernatants of several key mutants were characterized for the future matching of an expression host with a particular gene product. N-terminal sequencing of the five dominant proteins separated by SDS-PAGE from the culture supernatant was conducted. Two of the proteins identified were subtilisin-like proteinases and one was a pepsin-like proteinase. In addition, one protein was identified as an_-amylase and one remained unidentified. A 6.5 kb DNA fragment was isolated by Genomic Walking PCR using primers based on the _-amylase amino acid sequence. The amplified fragment contained the entire gene encoding_-amylase (amyl) and its regulatory sequences. Analysis showed that multiple transcripts were generated from the single _-amylase gene locus. / A series of expression vectors containg the _-amylase regulatory sequences and partial amyl gene were constructed. Several selection markers were screened and the hph gene conferring hygromycin resistance under the regulation of the Aspergillus nidulans gpd promoter was chosen and inserted into the amyl expression vectors. The gene encoding a red fluorescent protein DsRed-E5 was used as a reporter gene to test the expression system using mutant MQ.5.1 as host. However, no transformants were obtained by either biolistic transformation or protoplast transformation. Subsequently, an alternative strategy was developed using a thermostable xylanase B as a reporter. Thermostable xylanase activity was detected in the culture supernatants of several transformants. Production of xylanase by transformant SS41 which exhibited high secreted xylanase activity was investigated. Xylanase activity in the culture supernatant of SS41 was visualized by a zymogram gel assay. Two active proteins with molecular masses of around 27 and 30 kDA, which were larger than the predicted Mr of 25 kDA were detected. This is the first report describing successful expression of a recombinant thermostable bacterial enzyme in Ophiostoma. / Mode of access: World Wide Web. / 158 leaves col. ill

The respective kinetics of bacterial leucine aminopeptidase and [beta]-glucosidase activities were investigated to improve understanding of factors controlling activity and hydrolytic capacity in estuarine organic-poor sands. Depth distributions of enzyme activity and bulk organic matter content were determined in sediments of Aransas Bay and Copano Bay Texas, to investigate enzyme dynamics as related to the geochemical properties of the sediment. Vertical profiles of activity in sediment showed that the enzymes were more active at the surface and that the potential hydrolysis rate of leucine aminopeptidase was higher than that of [beta]-glucosidase. Vertical patterns of enzyme activity correlated (weakly) with variations in sediment organic matter (TOC, TN, and carbohydrates) content. Enrichments of sediment samples with monomeric organic compounds and inorganic nutrients did not affect leucine aminopeptidase and [beta]-glucosidase activities in short- and long-term incubations. Enzyme activity was independent of nutrient availability and suggested that microbial communities were not nutrient-limited. Time-course assays of bacterial hydrolysis of TOC, TN, and carbohydrates provided information about how substrate limitation may affect enzyme activity. Positive correlations between bulk TOC and TN content and enzyme activity indicated enzyme dependence on polymeric substrate content. Induction of enzyme activity after sediment enrichments with specific labile compounds confirmed the importance of available organic substrate to enzyme hydrolysis efficiency. A kinetic approach established the occurrence of enzyme inhibition and its effects on enzyme hydrolytic capacity. The addition of a specific-enzyme substrate to sediment samples modified enzyme parameters and indicated that a substrate-reversible type of inhibitor could reduce enzyme hydrolytic capacity. The addition of polyphenol, as a natural inhibitor of enzyme activity, to the sediment resulted in a concomitant reduction of leucine aminopeptidase activity and ammonium regeneration rate, and thus demonstrated a close coupling between enzyme activity and sediment ammonium regeneration. These research results demonstrate the dynamic nature of the hydrolytic enzymes, provide information about the mechanisms of induction and inhibition of activity, and demonstrate some implications of reducing the hydrolytic capacity to organic matter decomposition and nutrient regeneration rates. / text

Activity of enzymes was investigated in the soils of Norway spruce forests in the Bohemian Forest. The aim of the study was the determintation of the impact of temperature and plants dominant in understorey on the activity of extracellular enzymes in the soils in the watershed of Plešné and Čertovo Lake. The measurement of enzymes activities was conducted using the fluorometric method with model substrates.