Nitrogen oxides are targeted as important gas pollutants to be eliminated. Biological removal of nitrogen oxides, like the use of bio-trickling reactor, is gaining popularity over conventional methods. A bacterium isolated from a bio-trickling reactor showing high performance in removing nitrogen oxides was identified to be Pseudomonas mendocina. The genome of this isolate was sequenced using 454 pyrosequencing technology to a high level of completeness with 33 contigs and N50 contig length of 273 kbp. The genome was annotated by Prokaryotic Genome Automatic Annotaion Pipeline (PGAAP)and the strain was named P. mendocina DLHK. Examination of P. mendocina DLHK genome annotation found nitrate reductase but not nitrite reductase, nitric oxide reductase and nitrous oxide reductase. Novel genes or pathways might be available in P. mendocina DLHK contributingits denitrification function in the bio-trickling reactor. To better understand the evolution of Pseudomonas, a comprehensive comparative study was performed. Genomes of the Pseudomonasgenus were reannotated. Core genes were extracted to construct a phylogenomic tree using supermatrix approach. Effectiveness of using single phylogenetic marker in constructing phylogeny of Pseudomonas was evaluated using rpoB gene marker. Both methods generated phylogenetic trees of very similar topology, suggesting that rpoB gene can be a very effective marker in the rapid construction of Pseudomonas phylogeny.It is surprising to note that Azotobacter vinelandii DJ was included in the large clade of Pseudomonas and have the closest relationship with P. stutzeri in both phylogenetic trees, providing evidence that this species should be reclassified into the genus Pseudomonas. Cluster of Orthologous Groups (COG)category distribution of all pseudomonads were analysed for physiological significance. The large amount of gene categorised into the COG for amino acid metabolism and transcription may imply the importance for these 2 functions on the survival of pseudomonads. It is again surprising to note the COG distribution pattern of A. vinelandii DJ is different from other pseudomonads, especially to its closest phylogenetic neighbour P. stutzeri. Horizontal gene transfer events inpseudomonads were investigated because of its importance in prokaryotic evolution. The high congruence of the phylogemonic tree to most core genes suggests that the phylogenomic tree is a good piece of evidence describing the evolution of pseudomonads. It is a bit surprising to observe that quite a number of core genes may have exhibited horizontal gene transfer which show incongruence of the gene tree to the core genes. The impact of horizontal gene transfer events on the functionality and evolution of pseudomonads remains to be investigated. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Botany / Master / Master of Philosophy

published_or_final_version / abstract / toc / Botany / Master / Master of Philosophy

published_or_final_version / abstract / toc / Botany / Master / Master of Philosophy

In nature bacteria predominantly live on surfaces, in matrix-encased communities called biofilms. Biofilm formation displays dynamic developmental patterns resembling those of multicellular organisms. Using cooperative traits such as cell-cell signaling, bacteria in biofilms form complex architectures, known as microcolonies, in which cells become highly differentiated from their planktonic counterparts. Microcolonies are generally highly tolerant to bactericides, rendering biofilms extremely difficult to eradicate. The aim of this study was to investigate the last, and least understood stage of biofilm development, which involves the coordinated dispersal of single cells that revert to a free-swimming planktonic phenotype and escape from the biofilm. Strategies to induce biofilm dispersal are of interest due to their potential to prevent biofilms and biofilm-related infections. In the model organism Pseudomonas aeruginosa, reproducible patterns of cell death and dispersal can occur within biofilm structures, leaving behind empty or hollow microcolonies. These events were previously linked with the appearance of oxidative and/or nitrosative stress in mature microcolonies. Here, the involvement of reactive oxygen and nitrogen intermediates in biofilm development and dispersal processes was investigated in both mono- and mixed-species biofilms. By using specific fluorescent dyes and P. aeruginosa mutant strains, nitric oxide (NO), a by-product of anaerobic respiration and an important messenger molecule in biological systems, was found to play a major role in P. aeruginosa biofilm dispersal. Further, the results demonstrated that exposure to physiological, non-toxic concentrations of NO (in the low nanomolar range) causes biofilm dispersal in P. aeruginosa and restores its vulnerability to conventional antimicrobials. By using microarray techniques, NO was shown to induce global changes in genetic expression, including enhanced metabolic activity and motility and decreased adhesion and virulence in P. aeruginosa biofilms. The regulatory pathway implicated c-di-GMP, a newly discovered messenger molecule involved in the transition from sessility to motility in many bacterial species. NO-mediated dispersal was also observed in other single- and multi-species biofilms of clinically and industrially relevant organisms. Hence, the combined exposure to NO and bactericides was identified as a potential novel strategy for the removal of microbial communities, providing a low cost and environmentally safe solution to biofilm control.

In nature bacteria predominantly live on surfaces, in matrix-encased communities called biofilms. Biofilm formation displays dynamic developmental patterns resembling those of multicellular organisms. Using cooperative traits such as cell-cell signaling, bacteria in biofilms form complex architectures, known as microcolonies, in which cells become highly differentiated from their planktonic counterparts. Microcolonies are generally highly tolerant to bactericides, rendering biofilms extremely difficult to eradicate. The aim of this study was to investigate the last, and least understood stage of biofilm development, which involves the coordinated dispersal of single cells that revert to a free-swimming planktonic phenotype and escape from the biofilm. Strategies to induce biofilm dispersal are of interest due to their potential to prevent biofilms and biofilm-related infections. In the model organism Pseudomonas aeruginosa, reproducible patterns of cell death and dispersal can occur within biofilm structures, leaving behind empty or hollow microcolonies. These events were previously linked with the appearance of oxidative and/or nitrosative stress in mature microcolonies. Here, the involvement of reactive oxygen and nitrogen intermediates in biofilm development and dispersal processes was investigated in both mono- and mixed-species biofilms. By using specific fluorescent dyes and P. aeruginosa mutant strains, nitric oxide (NO), a by-product of anaerobic respiration and an important messenger molecule in biological systems, was found to play a major role in P. aeruginosa biofilm dispersal. Further, the results demonstrated that exposure to physiological, non-toxic concentrations of NO (in the low nanomolar range) causes biofilm dispersal in P. aeruginosa and restores its vulnerability to conventional antimicrobials. By using microarray techniques, NO was shown to induce global changes in genetic expression, including enhanced metabolic activity and motility and decreased adhesion and virulence in P. aeruginosa biofilms. The regulatory pathway implicated c-di-GMP, a newly discovered messenger molecule involved in the transition from sessility to motility in many bacterial species. NO-mediated dispersal was also observed in other single- and multi-species biofilms of clinically and industrially relevant organisms. Hence, the combined exposure to NO and bactericides was identified as a potential novel strategy for the removal of microbial communities, providing a low cost and environmentally safe solution to biofilm control.

Bibliography: leaves 207-220. Pseudomonas corrigata strain 2140 (Pc2140), isolated from wheat field soil in Australia, antagonises the take-all fungus, Gaeumannomyces graminis var. tritici (Ggt) in vitro and significantly reduces take-all symptoms on wheat in pot trials. This study investigates the mechanisms by which the biocontrol agent reduces the disease symptoms. Biochemical analysis of metabolites of P. corrugata 2140 reveal a number of compounds potentially antagonistic to Ggt and which may play a role in disease control. These include water-soluble antibiotics, siderophores, proteases, peptides and volatiles including hydrogen cyanide.

Bibliography: leaves 207-220. / 220 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Pseudomonas corrigata strain 2140 (Pc2140), isolated from wheat field soil in Australia, antagonises the take-all fungus, Gaeumannomyces graminis var. tritici (Ggt) in vitro and significantly reduces take-all symptoms on wheat in pot trials. This study investigates the mechanisms by which the biocontrol agent reduces the disease symptoms. Biochemical analysis of metabolites of P. corrugata 2140 reveal a number of compounds potentially antagonistic to Ggt and which may play a role in disease control. These include water-soluble antibiotics, siderophores, proteases, peptides and volatiles including hydrogen cyanide. / Thesis (Ph.D.)--University of Adelaide, Dept. of Crop Protection, 1996