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Protein recycling in Bering Sea Algal incubations

ABSTRACT: 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.

INSTRUMENT(S): Finnigan MAT instrument model

ORGANISM(S): Thalassiosira Pseudonana

TISSUE(S): Cell Suspension Culture

SUBMITTER: Brook Nunn

LAB HEAD: Brook L. Nunn

PROVIDER: PXD006417 | Pride | 2017-05-19

REPOSITORIES: Pride

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			Action	DRS
	4ABCDE_Full_mz.prot.xls	Xls
	Oct_12_2009_Nunn_Bering_Sea_40.mzXML	Mzxml
	Oct_12_2009_Nunn_Bering_Sea_40.raw	Raw
	Oct_12_2009_Nunn_Bering_Sea_41.mzXML	Mzxml
	Oct_12_2009_Nunn_Bering_Sea_41.raw	Raw

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Project description:Peptides were identified during degradation of the diatom Thalassiosira weissflogii with and without the use of the enzyme trypsin. This allows comparison of protein degradation occurring within the experiment (no trypsin added; peptides already present due to in situ degradation) and the protein still available for future degradation (peptides released from protein when trypsin is added during analysis). Over the 12-day degradation experiment 31% of the particulate organic carbon was depleted and there was no preferential degradation of the overall protein pool. However, there was distinct differentiation in the cellular location, secondary structure and modifications of the peptides that were either degraded or remained. During the initial period of rapid algal decay and bacterial growth, intracellular components from the cytoplasm were selectively consumed, resulting in the accumulation of membrane-associated proteins and peptides in the detrital pool. Accompanying this was an increase in the importance of membrane-bound ɑ-helix motifs. At the end of the 12 day experiment, the natural bacterial assemblage was focusing on degradation of membrane-associated proteins, which was the dominant source of peptides in both the residual pool (released by trypsin) and the actively degraded pool (no trypsin added). Methylated arginine, a post-translationally modified amino acid that is produced within the diatom prior to senescence, is found in high amounts within the detrital peptide pool, suggesting a link between in-cell modification and resistance to immediate degradation. Another modification - asparagine deamidation - appears to occur during degradation and deamidated peptides also accumulate within the detritus. The bacterial community decomposing the algal material was rich in proteobacteria, and employed a growth approach focusing on accumulation of solubilized material across their membranes and on DNA replication. At this early stage of diagenesis, no changes in bulk amino acids (THAA) were observed, yet a peptidomic approach allowed us to observe the differential changes in diatom protein preservation by discriminating between intracellular location, secondary structure, and modifications status.

Project description:Pelagic aggregates function as hotspots for microbial activity and biological carbon pumps for exporting OM fixed by photoautotrophs to sediments in lakes and oceans. In iron-rich (ferruginous) lakes, photoferrotrophic or chemolithoautotrophic bacteria appear to contribute to CO2 fixation by oxidizing reduced iron which leads to the formation of iron-rich pelagic aggregates called iron-snow. In acidic lakes, iron snow is colonized mainly by acidophilic iron-cycling microbes that can trigger interspecies aggregation mechanisms. However, the significance of iron oxidizers in carbon fixation, their general role in iron snow functioning, and the flow of carbon within iron snow is still unclear. Here, we combined a two-year metatranscriptome analysis with a 13CO2 metabolic labeling approach to determine general metabolic activities. Protein-based stable isotope probing (protein-SIP) was used to trace the 13CO2 incorporation in iron snow microcosms over time under both oxic and anoxic conditions. Analysis of our mRNA-derived metatranscriptome data identified four key players (Leptospirillum, Ferrovum, Acidithrix, Acidiphilium) with relative abundances (59.6%-85.7%) in iron snow encoding a variety of ecologically relevant pathways, including carbon fixation, polysaccharide biosynthesis, and flagellar-based motility. We did not detect transcriptional activity for carbon fixation from archaea or eukaryotes. The largest numbers of expressed genes (3008, 2991, 2936) matched to the genomes of our previously obtained iron snow isolates (Acidithrix sp. C25, Acidiphilium sp. C61, Acidocella sp. C78) separately. 13CO2 incorporation studies identified Leptospirillum and Ferrovum, as the main active chemolithoautotrophs under oxic conditions, and Ferrovum was the main active organism under anoxic conditions as well. Small amounts of labeled 13C (Relative isotope abundance: 1.0%-5.3%) were found in the heterotrophic Acidiphilium and Acidocella. Overall, our data show that iron oxidizers play an important role in the formation of iron minerals and CO2 fixation, but the majority of fixed C apparently did not reach other iron snow microbes. This finding suggests that most of the fixed C will be directly exported to the sediment without feeding heterotrophs in the water column in acidic ferruginous lakes.

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Protein recycling in Bering Sea Algal incubations

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