Despite being abundant in the earth's crust, the concentration of Fe in many oceanic regions is so low that it is limiting to the growth of photosynthetic plankton. Heterotrophic bacteria play key roles in the oceanic cycling of carbon and nutrients, but it is unclear whether they can be Fe-deficient in nature, or what possible effects Fe-deficiency might have on their ecology and physiology. In chapter 1, I investigated the response of a natural bacterial community to a mesoscale Fe-enrichment experiment in the northeast subarctic Pacific. The addition of Fe to surface waters caused a rapid stimulation of bacterial growth and production, and induced the organic Fe uptake systems of bacteria. These findings suggest that bacteria responded directly to increased Fe availability, and may be Fe-deficient in situ. In chapter 2, I examined the effects of Fe-deficiency on the coupled processes of carbon catabolism and adenosine triphosphate (ATP) production in cultures of the marine bacterium Pseudoalteromonas haloplanktis. In Fe-limited cells, Fe-dependent oxidative pathways of ATP production were downregulated, leading to an intracellular energy deficit. Thus, by altering carbon metabolism and energy acquisition of heterotrophic bacteria, Fe may affect the cycling of carbon in parts of the sea.

Batch cultures of Candidatus Pelagibacter ubique were grown under iron-, organosulfur-, and nitrogen-limiting conditions to understand how this ubiquitous marine bacterium responds to and interacts with environments where growth is limited by the availability of these nutrients. Global gene expression was monitored using microarrays and quantitative mass spectrometry to observe both transcriptional and post-transcriptional responses to nutrient limitation. Iron- and nitrogen-limited cultures were characterized by increased transcription and translation of transporters involved in acquisition of the limiting nutrient, whereas organosulfur-limited cultures were not. Methionine synthesis genes downstream of S-adenosyl methionine riboswitches were up-regulated in mRNA and protein during organosulfur-replete stationary phase. Comparative genomics also revealed Ca. Pelagibacter to be the only genus among the free-living Alphaproteobacteria to lack a P[subscript II]-mediated nitrogen regulatory pathway – a pathway which may be complemented in Ca. P. ubique by putative riboswitches and a citric acid cycle able to bypass the glutamate precursor 2-oxoglutarate. Overall, the results of this study provide insight into the regulatory and metabolic processes of this ecologically significant organism, and enable better interpretation of metatranscriptomic and meta-proteomic surveys by identifying sfuC and amtB as likely biomarkers for iron and nitrogen limitation, respectively, in natural Ca. P. ubique populations. / Graduation date: 2012 / Access restricted to the OSU Community at author's request from Dec. 13, 2011 - Dec. 13, 2013

Ultraviolet radiation reaching the Earth’s surface (UVR, 280-400 nm) may penetrate deep into the clear oligotrophic waters influencing a large part of the euphotic layer. Marine heterotrophic bacteria at the surface of the oceans are especially sensitive to the damaging solar radiation due to their haploid genome with little or no functional redundancy and lack of protective pigmentation. In a context of climate change and ozone depletion, it is clearly important to understand the physiology and underlying molecular UVR responses of abundant marine bacteria species. We chose the marine ultramicrobacterium Sphingopyxis alaskensis as a reference species to study the impact of solar radiation due to its numerical abundance in oligotrophic waters and its photoresistance, previously reported. For this purpose, we focused on the formation of the two major UVB-induced DNA photoproducts (CPDs and 6-4PPs) as well as the differential protein expression under solar radiation. We first demonstrated that the GC content of prokaryotic genome had a major effect on the formation of UVB-induced photoproducts, quantified by HPLC-MS/MS. Due to its high GC content, S. alaskensis presented a favoured formation of highly mutagenic cytosine-containing photoproducts and therefore would be more susceptible to UVinduced mutagenesis. By comparing S. alaskensis to another marine bacterium Photobacterium angustum, we observed for the latter strain a remarkable resistance to high UVB doses associated with a decrease in the rate of formation of CPDs explained by a non-conventional activity of photolyase. We also demonstrated that DNA damage in S. alaskensis was markedly modulated by growth temperature and time spent in stationary phase. In order to assess the effects that environmental UV-R had on regulatory networks and pathways of S. alaskensis, and determine how the cell’s physiology was affected, a quantitative proteomics investigation was performed. Changes in proteome were analyzed, with the recent and powerful mass spectrometry based approach using iTRAQ methodology. Approximately, one third of the proteome of S. alaskensis was identified, with 119 statistically and significantly differentially abundant proteins. Cellular processes, pathways and interaction networks were determined and gave us unique insight into the biology of UV response and adaptation of S. alaskensis.

The marine bacterium Sphingopyxis alaskensis was isolated as one of the most numerically abundant bacteria from cold (4–10??C) nutrient depleted waters in the North Pacific Ocean. The objective of this study was to examine cold adaptation of S. alaskensis by using proteomics to examine changes in global protein levels caused by growth at low (10??C) and high (30??C) temperatures. Stable isotope labelling-based quantitative proteomics was used, and a rigorous post-experimental data processing workflow adapted from microarray-based methods was developed. The approach included metabolic labelling with 14N/15N and normalisation and statistical testing of quantitative proteomics data. Approximately 400,000 tandem mass spectra were generated resulting in the confident identification of 2,135 proteins (66% genome coverage) and the quantitation of 1,172 proteins (37% genome coverage). Normalisation approaches were evaluated using cultures grown at 30??C and labelled with 14N and 15N. For 10??C vs. 30??C experiments, protein quantities were normalised within each experiment using a multivariate lowess approach. Statistical significance was assessed by combining data from all experiments and applying a moderated t-test using the empirical Bayes method with the limma package in R. Proteins were ranked after calculating the B-statistic and the Storey-Tibshirani false discovery rate. 217 proteins (6% genome coverage) were determined to have significant quantitative differences. In achieving these outcomes a range of factors that impact on quantitative proteomics data quality were broadly assessed, resulting in the development of a robust approach that is generally applicable to quantitative proteomics of biological system. The significantly differentially abundant proteins from the proteomics data provided insight into molecular mechanisms of cold adaptation in S. alaskensis. Important aspects of cold adaptation included cell membrane restructuring, exopolysaccharide biosynthesis, lipid degradation, carbohydrate and amino acid metabolism, and increased capacity of transcriptional and translational processes. A number of cold adaptive responses in S. alaskensis were novel, including a specific cold-active protein folding pathway, a possible thermally-controlled stringent response, and biosynthesis of intracellular polyhydroxyalkanoate reserve material. The overall study provided important new insight into the evolution of growth strategies necessary for the effective competition of S. alaskensis in cold, oligotrophic environments.

The marine bacterium Sphingopyxis alaskensis was isolated as one of the most numerically abundant bacteria from cold (4–10??C) nutrient depleted waters in the North Pacific Ocean. The objective of this study was to examine cold adaptation of S. alaskensis by using proteomics to examine changes in global protein levels caused by growth at low (10??C) and high (30??C) temperatures. Stable isotope labelling-based quantitative proteomics was used, and a rigorous post-experimental data processing workflow adapted from microarray-based methods was developed. The approach included metabolic labelling with 14N/15N and normalisation and statistical testing of quantitative proteomics data. Approximately 400,000 tandem mass spectra were generated resulting in the confident identification of 2,135 proteins (66% genome coverage) and the quantitation of 1,172 proteins (37% genome coverage). Normalisation approaches were evaluated using cultures grown at 30??C and labelled with 14N and 15N. For 10??C vs. 30??C experiments, protein quantities were normalised within each experiment using a multivariate lowess approach. Statistical significance was assessed by combining data from all experiments and applying a moderated t-test using the empirical Bayes method with the limma package in R. Proteins were ranked after calculating the B-statistic and the Storey-Tibshirani false discovery rate. 217 proteins (6% genome coverage) were determined to have significant quantitative differences. In achieving these outcomes a range of factors that impact on quantitative proteomics data quality were broadly assessed, resulting in the development of a robust approach that is generally applicable to quantitative proteomics of biological system. The significantly differentially abundant proteins from the proteomics data provided insight into molecular mechanisms of cold adaptation in S. alaskensis. Important aspects of cold adaptation included cell membrane restructuring, exopolysaccharide biosynthesis, lipid degradation, carbohydrate and amino acid metabolism, and increased capacity of transcriptional and translational processes. A number of cold adaptive responses in S. alaskensis were novel, including a specific cold-active protein folding pathway, a possible thermally-controlled stringent response, and biosynthesis of intracellular polyhydroxyalkanoate reserve material. The overall study provided important new insight into the evolution of growth strategies necessary for the effective competition of S. alaskensis in cold, oligotrophic environments.

Ultraviolet radiation reaching the Earth’s surface (UVR, 280-400 nm) may penetrate deep into the clear oligotrophic waters influencing a large part of the euphotic layer. Marine heterotrophic bacteria at the surface of the oceans are especially sensitive to the damaging solar radiation due to their haploid genome with little or no functional redundancy and lack of protective pigmentation. In a context of climate change and ozone depletion, it is clearly important to understand the physiology and underlying molecular UVR responses of abundant marine bacteria species. We chose the marine ultramicrobacterium Sphingopyxis alaskensis as a reference species to study the impact of solar radiation due to its numerical abundance in oligotrophic waters and its photoresistance, previously reported. For this purpose, we focused on the formation of the two major UVB-induced DNA photoproducts (CPDs and 6-4PPs) as well as the differential protein expression under solar radiation. We first demonstrated that the GC content of prokaryotic genome had a major effect on the formation of UVB-induced photoproducts, quantified by HPLC-MS/MS. Due to its high GC content, S. alaskensis presented a favoured formation of highly mutagenic cytosine-containing photoproducts and therefore would be more susceptible to UVinduced mutagenesis. By comparing S. alaskensis to another marine bacterium Photobacterium angustum, we observed for the latter strain a remarkable resistance to high UVB doses associated with a decrease in the rate of formation of CPDs explained by a non-conventional activity of photolyase. We also demonstrated that DNA damage in S. alaskensis was markedly modulated by growth temperature and time spent in stationary phase. In order to assess the effects that environmental UV-R had on regulatory networks and pathways of S. alaskensis, and determine how the cell’s physiology was affected, a quantitative proteomics investigation was performed. Changes in proteome were analyzed, with the recent and powerful mass spectrometry based approach using iTRAQ methodology. Approximately, one third of the proteome of S. alaskensis was identified, with 119 statistically and significantly differentially abundant proteins. Cellular processes, pathways and interaction networks were determined and gave us unique insight into the biology of UV response and adaptation of S. alaskensis.

Thesis (Ph. D.)--University of California, San Diego, 1998. / Vita. Includes bibliographical references (p. 122-138).

Thesis (M.Sc.)--Memorial University of Newfoundland, 2003. / Includes bibliographical references. Also available online.

No description available.

Though this current study was initiated independently and was not a test laboratory for the taxonomic sub-committee's evaluative program, the problem outlined in this treatise was also designed in an effort to test certain characteristics of the actinomycetes of both a biochemical and morphological nature. This problem employed methods that might absolve or establish certain criteria for taxonomic use in the group of actinomycetes.