Epiphytic bacterial communities on the surfaces of marine algae are poorly characterised. Most information available on marine bacterial epiphytes is derived from culture-based studies. With the rapid development of molecular community analysis technologies, it is now possible to obtain a more comprehensive picture of marine microbial populations on living surfaces. The intertidal macroalga Ulva australis, belongs to the cosmopolitan group of green marine algae (Ulvales) known to require the presence of bacteria for normal growth and has been suggested to employ specific bacteria for the defence against fouling by micro- and macro-organisms. This thesis has examined the composition and structure of the surface associated bacterial community on Ulva australis using 16S rRNA gene clone library, denaturing gradient gel electrophoresis (DGGE), and catalysed reporter deposition ??? fluorescence in situ hybridisation (CARD-FISH) analysis. The 16S rRNA gene clone library revealed that the five main bacterial groups present in the surface associated community were Bacteriodetes, Planctomycetes, Alpha-, Gamma-, and Delta-Proteobacteria. Twenty-two sequence phylotypes were identified, suggesting that the epiphytic community was of relatively low diversity. A clone similar to an algal morphogenesis inducing Cytophaga strain was identified, indicating that U. australis harbours bacteria important for thallus structural maintenance. DGGE analysis showed that while the bacterial community varied over spatial and temporal (seasons) scales it also included a stable subpopulation consistently associated with the seaweed surface. Sequencing of selected DGGE bands suggested that members of the Alphaproteobacteria and the Bacteriodetes belonged to the stable subpopulation. Using CARD-FISH with different phylogenetic probes demonstrated that Alphaproteobacteria (~ 70%) and Cytophaga-Flavobacteria (~13%) constituted the majority of bacterial cells on the surface of U. australis. A comparison of the results provided by the molecular community analysis methods, employed in this thesis, and those of culturing of epiphytic bacteria from U. australis revealed that each approach provides different patterns of phylogeny and extent of diversity. For example, the culture collection and the clone library detected a relatively high amount of Gammaproteobacteria, however, DGGE and CARD-FISH did not. Also, low species diversity clone sequences and isolates of Alphaproteobacteria contrasted with the high numbers detected by the DGGE analysis. In addition to the phylogentic determination of the epiphytic bacterial community, CARDFISH was also used to assess the organisation and distribution of bacterial cells across different zonal regions on seaweed surface. It was found that approximately 40% of bacterial cells clustered in aggregates, or microcolonies. These aggregations were considered to be heterogeneous in composition and were mainly comprised of multiply species. The occurrence of more non-viable solitary single rather than aggregated cells suggests that aggregates might offer greater protection to bacterial cells from the harsh conditions in the intertidal zone. More broadly, CARD-FISH was found to be a useful tool for studying microcolonies and was also successfully applied to detect slow growing soil microcolonies cultivated using a novel soil substrate membrane system culturing technique without the need to perform an rRNA enrichment incubation. The findings in this thesis, as described from the application of a number of molecular community analysis techniques such as clone library, DGGE and CARD-FISH, have improved our understanding of the diversity and structure of the epiphytic bacterial community associated with U. australis. Morevover, the information provided may to design future studies in the ecology of bacteria-seaweed interactions, including symbiotic interactions, and aid in marine biotechnology applications such as identifying bacteria which produce bioactive secondary metabolites.

The dark-green pigmented marine bacterium Pseudoalteromonas tunicata produces several extracellular compounds against a range of common fouling organisms including bacteria, fungi, protozoa, diatoms, invertebrate larvae and algal spores. The regulator WmpR, which has N-terminal similarity to ToxR from Vibrio cholerae and CadC from Escherichia coli, controls all of the pigment and antifouling phenotypes. These compounds appear at the onset of stationary phase. The role of WmpR as a stationary phase regulator in P. tunicata was investigated in this thesis. Starvation and stress studies demonstrated that WmpR does not appear to control genes necessary for survival during carbon, phosphate or nitrogen starvation and UV/hydrogen peroxide stress. Intriguingly, phosphate starvation caused pigmentation of wmpR mutant (D2W2) logarithmic phase cells, suggesting a second regulation of the pigments (and thus antifouling compounds) that could be mediated by the PhoR/B twocomponent regulatory system. Proteomic analysis using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) found that 11 proteins were differentially regulated by WmpR, and the identities of some of these proteins suggested a role for WmpR as a general stationary phase regulator rather than a specific starvation or stress regulator. Gene expression studies using RNA-arbitrarily primed PCR introduced a new role for WmpR as a regulator of iron acquisition; a TonB-dependant outer membrane receptor gene and a non-ribosomal peptide synthetase (NRPS) gene were up-regulated in the stationary phase Wt strain compared to the D2W2 strain. An assay for iron-binding activity supported the proposal that the NRPS may be making a siderophore. Further studies demonstrated that WmpR is required for survival under long-term low-iron conditions and that the pigments and antifouling genes are down-regulated during low-iron, while biofilm formation is up-regulated. WmpR also appears to constitutively regulate the production of iron-binding compounds, a novel regulation of iron acquisition that has not been seen in other organisms studied so far. A model is proposed that describes WmpR as responding to environmental signals, including iron, and co-ordinating the expression of a complex regulon including a number of genes involved in iron acquisition, general stationary phase physiology and bioactive secondary metabolite production.

Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisor: David L. Kirchman, College of Marine and Earth Studies. Includes bibliographical references.

Thesis (Ph. D.)--University of Delaware, 2006. / Principal faculty advisor: David L. Kirchman, College of Marine and Earth Studies. Includes bibliographical references.

Graduation date: 1992

Many bacterial diseases were controlled by antibiotics since mid 1900s. However, over use of the drugs leads to the prevalence of resistant strains, some of them are resistant to essentially all of the commercial antibiotics except for one or two. Therefore, new drugs are needed to combat the die-hard resistant pathogens. This study is aimed at isolating bacteria with antimicrobial activities from the intertidal soils of Taiwan. A total of 25 samples were collected from 5 locations, including Peng-Hu, Da-Peng Bay, Ken-ding, Gueishan Island and Little Liu-chiu. Marine Agar 2216 and Actinomycete Isolation Agar were used for cultivation. Of 313 bacterial isolated, 47 of them showed antimicrobial activities against at least one of the 7 indicators (Bacillus cereus, Staphylococcus aureus, Klebsiella pneumonia, Salmonella typhimurium, Pseudomonas aeruginosa, Vibrio harveyi, Escherichia coli). The 47 strains were classified based on 16S rRNA phylogeny. The majority of them are Pseudoalteromonas spp. (31) and Bacillus spp. (9) as well as Virgibacillus sp. (1), Vibrio sp. (1), Streptomyces sp. (1) and Microbulbifer spp. (4). Over all, 10/47 are Gram positive and 37/47 require added salts for growth. The titers of antimicrobial substances as judged by ethyl acetate extraction were influenced by cultivation conditions, such as: growth temperature, types of media and time.

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Microbiology and Molecular Genetics." Includes bibliographical references (p. 89-98).

Thesis (M.S.)--University of Hawaii at Manoa, 2005. / Includes bibliographical references (leaves 160-171). Also available via World Wide Web.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (p. 146-162).

Because of the eutrophication resulting from increasing anthropogenic activities, hypoxia (i.e. dissolved oxygen < 2.8 mg O2 L-1) is on the rise globally. The objective of this research was to understand more about the effects of hypoxia on the marine benthic communities. Particularly, it focused on the latent effects and indirect effects of hypoxia by investigating how early exposure to hypoxia affect the later life stage of a marine gastropod Crepidula onyx, and how hypoxia alter the bacterial composition of biofilms and the subsequent larval settlement of marine invertebrates. In the first study, the larvae of C. onyx were exposed to 2, 3, and 6 mg O2 l-1. Under low food concentration (Isochrysis galbana at 1 × 105 cells l-1), larvae in both hypoxic treatments (2 and 3 mg O2 l-1) required a longer time to become competent to metamorphose. But when they did, they had a similar size and total lipid content to the control larvae. Moreover, the latent effects of early hypoxic exposure on the juvenile growth were evident. After 2 weeks development in field, the growth rate, mean dry weight and filtration rate of juveniles were significantly reduced in the hypoxic treatments. However, there was no discernible effect on larvae or juveniles when the food concentration during the larval stage was doubled (I. galbana at 2 × 105 cells l-1), suggesting that the latent effects of hypoxia can be offset by larval access to high algal concentration. In the second study, the biofilms were exposed to hypoxia and normoxia in microcosms for up to 7 days, and their bacterial community composition was analysed by terminal-restriction fragment length polymorphism (T-RFLP). The results suggested that hypoxia altered the bacterial community structure within biofilms, and the difference between the hypoxia and normoxia treatments increased through the length of exposure period. The resulting changes in biofilms did not alter the larval settlement response of a model species (i.e. C. onyx) in laboratory assays. Nevertheless, when the biofilms were deployed in the field to allow natural larval settlement and recruitment, biofilms that had been exposed to hypoxia altered the overall larval settlement pattern of different marine invertebrates, potentially leading to a shift in the benthic invertebrate community. This research suggested that periodic hypoxic events and the resulting exposure of organisms to hypoxia during their early development might have effects that persist across the life history. Moreover, it highlighted the possibility that the effects of hypoxia on species composition and structure of benthic invertebrate communities might be mediated through changes in biofilms and subsequently larval settlement and recruitment. To conclude, this research demonstrated that hypoxia could affect the growth in the later life stages of marine invertebrates and the recruitment of the benthic communities. / published_or_final_version / Biological Sciences / Master / Master of Philosophy