Inter-Laboratory Comparison of LC-MS analysis of algal and marine DOM - Boiteau
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Synapt G2 MS
ORGANISM(S): Algal Lysate
SUBMITTER: 
Rene Boiteau
PROVIDER: MSV000088207 | MassIVE | Fri Oct 08 16:04:00 BST 2021
REPOSITORIES: MassIVE
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Project description:LC-MS analysis of algal and marine DOM at Oregon State University (Boiteau Lab) as part of the "Inter-Laboratory Comparison of LC-MS analysis of algal and marine DOM" study by the Inter-Lab LC-MS/MS Consortium
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Project description:Unicellular algae, termed phytoplankton, greatly impact the marine environment by serving as the basis of marine food webs and by playing central roles in biogeochemical cycling of elements. The interactions between phytoplankton and heterotrophic bacteria affect the fitness of both partners. It is becoming increasingly understood that metabolic exchange determines the nature of such interactions, but the underlying molecular mechanisms remain underexplored. Here, we investigated the molecular and metabolic basis for the bacterial lifestyle switch, from coexistence to pathogenicity, in Sulfitobacter D7 during interactions with Emiliania huxleyi, a cosmopolitan bloom-forming phytoplankter. The interaction displays two distinct phases: first, there is a coexisting phase in which the alga grows exponentially and the bacterium grows as well. The interaction shifts to pathogenic when the virulence of Sulfitobacter D7 towards E. huxleyi is invoked upon exposure to high concentrations of algal dimethylsulfoniopropionate (DMSP), which occurs when the algae reach stationary growth or when DMSP is applied exogenously to algae in exponential growth. We aimed to unravel the response of Sulfitobacter D7 to the pathogenicity-inducing compound, DMSP, and to different algae-derived infochemicals that affect the lifestyle of the bacterium. We grew Sulfitobacter D7 in conditioned media (CM) derived from algal cultures at the different growth phases, exponential and stationary (Exp-CM and Stat-CM, respectively), in which DMSP concentration is low and high, respectively. This enabled us to separate between different phases of the interaction with E. huxleyi, i.e., Exp-CM representing the coexisting phase, and Stat-CM representing the pathogenic phase. An additional pathogenicity-inducing treatment was Exp-CM supplemented with 100 µM DMSP (herein Exp-CM+DMSP). This condition mimicked co-cultures to which we added DMSP exogenously and thus induced Sulfitobacter D7 pathogenicity, which lead to death of exponentially growing E. huxleyi. In order to identify bacterial genes that are specifically responsive to DMSP, and are not affected by other algae-derived factors, we grew Sulfitobacter D7 in defined minimal medium (MM), lacking algal metabolites, supplemented with 100 µM DMSP (herein MM+DMSP), and examined the transcriptional response. After 24 h of Sulfitobacter D7 growth in all 5 media, triplicates were taken for transcriptomic analysis. Altogether, this experimental design allowed to expand our understanding on the bacterial response to DMSP, algal infochemicals and which of these are essential for coexistence and pathogenicity.
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Project description:Protein present in phytoplankton represents a large fraction of the organic nitrogen and carbon transported from its synthesis in surface waters to marine sediments. Yet relatively little is known about the longevity of identifiable protein in situ, or the potential modifications to proteins that occur during bloom termination, protein recycling and degradation. To address this knowledge gap, diatom-dominated phytoplankton was collected during the Bering Sea spring blooms of 2009 and 2010, and incubated under darkness in separate shipboard degradation ex periments spanning 11 and 53 d, respectively. In each experiment, the protein distribution was monited over time using shotgun proteomics, along with total hydrolyzable amino acids (THAAs), total protein, particulate organic carbon (POC) and nitrogen (PN), and bacterial cell abundance. Identifiable proteins, total protein and THAAs were rapidly lost during the first 5 d of enclosure in darkness in both incubations. Thereafter the loss rate was slower, and it declined further after 22 d. The initial loss of identifiable biosynthetic, glycolysis, metabolism and translation proteins after 12 h may represent shutdown of cellular activity among algal cells. Additional peptides with glycan modifications were identified in early incubation time points, suggesting that such protein modifications could be used as a marker for internal recycling processes and possibly cell death. Protein recycling was not uniform, with a subset of algal proteins including fucoxanthin chlorophyll binding proteins and RuBisCO identified after 53 d of degradation. Non-metric multidimensional scaling was used to compare the incubations with previous environmental results. The results confirmed recent observations that some fraction of algal proteins can survive water column recycling and undergo transport to marine sediments, thus contributing organic nitrogen to the benthos.
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