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Quantitative contributions of cyanobacterial alkaline phosphatases to biogeochemical rates in the subtropical North Atlantic

ABSTRACT: Microbial enzymes alter marine biogeochemical cycles by catalyzing chemical transformations that bring elements into and out of particulate organic pools. These processes are traditionally studied through enzyme rate-based estimates and nutrient-amendment bioassays, but these approaches lack the ability to resolve species-level contributions to enzymatic rates. Molecular methods including proteomics have the potential to link the contributions of specific populations to the overall community enzymatic rate; this is important because organisms will have distinct enzyme characteristics, feedbacks, and responses to perturbations. Integrating molecular methods with rate measurements can be achieved quantitatively through absolute quantitative proteomics. Here, we use the subtropical North Atlantic as a model system to probe how absolute quantitative proteomics can provide a more comprehensive understanding of nutrient limitation in marine environments. The experimental system is characterized by phosphorus stress and potential metal-phosphorus co-limitation due to dependence of the organic phosphorus scavenging enzyme alkaline phosphatase on metal cofactors; we performed nutrient amendment incubation experiments to investigate how alkaline phosphatase abundance and activity is affected by trace metal additions. We show that the two most abundant picocyanobacteria, Prochlorocccus and Synechococcus are minor contributors to total alkaline phosphatase activity as assessed by a widely used enzyme assay. This was true even when trace metals were added, despite both species having the genetic potential to utilize both the Fe and Zn containing enzymes, PhoX and PhoA respectively. Serendipitously, we also found that the alkaline phosphatases responded to cobalt additions suggesting possible substitution of the metal center by Co in natural populations of Prochloroccocus (substitution for Fe in PhoX) and Synechococcus (substitution for Zn in PhoA). This integrated approach allows for a nuanced interpretation of how nutrient limitation affects marine biogeochemical cycles and highlights the benefit of building quantitative connections between rate and “-omics” based measurements.

INSTRUMENT(S):

ORGANISM(S): Prochlorococcus Marinus Subsp. Pastoris Environmental Samples <cyanobacteria

SUBMITTER: Noelle Held

LAB HEAD: Noelle Held

PROVIDER: PXD053717 | Pride | 2025-10-03

REPOSITORIES: Pride

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			Action	DRS
	190405_1D_NAH_Koko_Stn2AA_10fmol_Ziploc1_BATSv6.raw	Raw
	190405_1D_NAH_Koko_Stn2AB_10fmol_Ziploc1_BATSv6.raw	Raw
	190405_1D_NAH_Koko_Stn2BA_10fmol_Ziploc1_BATSv6.raw	Raw
	190405_1D_NAH_Koko_Stn2BB_10fmol_Ziploc1_BATSv6.raw	Raw
	190405_1D_NAH_Koko_Stn2CA_10fmol_Ziploc1_BATSv6.raw	Raw

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Project description:Relatively little is known about the presence and regulation of pathways involved in nutrient acquisition in the brown tide forming alga, Aureococcus anophagefferens. In this study, Long-SAGE (Serial Analysis of Gene Expression) was used to profile the A. anophagefferens transcriptome under nutrient replete (control), and nitrogen (N) and phosphorus (P) deficiency with the goal of understanding how this organism responds at the transcriptional level to varying nutrient conditions. This approach has aided A. anophagefferens genome annotation efforts and identified a suite of genes up-regulated by N and P deficiency, some of which have known roles in nutrient metabolism. Genes up-regulated under N deficiency include an ammonium transporter, an acetamidase/formamidase, and two peptidases. This suggests an ability to utilize reduced N compounds and dissolved organic nitrogen, supporting the hypothesized importance of these N sources in A. anophagefferens bloom formation. There are also a broad suite of P-regulated genes, including an alkaline phosphatase, and two 5’-nucleotidases, suggesting A. anophagefferens may use dissolved organic phosphorus under low phosphate conditions. These N- and P-regulated genes may be important targets for exploring nutrient controls on bloom formation in field populations. Aureococcus anophagefferens CCMP 1984 was obtained from the Provasoli-Guillard Center for the Culture of Marine Phytoplankton (CCMP). The cultures were grown at 18C on a 14 h:10 h light:dark cycle (140 µmol quanta m-2 s-1). Nitrogen- and phosphate-replete (883 µM NO3- and 36.3 µM PO43-) cells, –N (40 µM NO3-) cells, and –P (1 µM PO43-) cells were grown in autoclaved L1 media with no Si (Guillard and Hargraves 1993), prepared using 0.2 µm filtered Vineyard Sound seawater. Vitamins (thiamine, biotin, and B12) were sterile filtered and added to the media after autoclaving. Replete cells were harvested during mid log phase of growth, while –N and –P cells were harvested at the onset of stationary phase when N or P was depleted.

Project description:Phosphorus (P) is a critical driver of phytoplankton growth and ecosystem structure and function in the ocean. Diatoms are an abundant and widespread functional group of phytoplankton that are responsible for significant amounts of primary production in the ocean, however there has not been a comprehensive study of diatom physiological responses to P deficiency. Here, we coupled deep sequencing of transcript tags and quantitative proteomic analysis from the diatom Thalassiosira pseudonana grown under P-replete and P-deficient conditions. The reads (tags) were mapped to the T. pseudonana genome sequence, confirming expression of 91% of the modeled gene set. A total of 318 genes were differentially regulated with a false discovery rate of p<0.05. A total of 1264 proteins were detected, and of those 136 were differentially expressed with a false discovery rate of p<0.05. Significant changes in the abundance of transcripts and proteins were observed and these changes were coordinated for glycolysis, translation, and multiple biochemical responses to P deficiency. These data demonstrate that diatom P deficiency results in changes in cellular P allocation through polyphosphate production, increased P transport, a switch to utilization of dissolved organic P (DOP) through increased production of alkaline phosphatase metalloenzymes and a diesterase, and a remodeling of the cell surface through production of sulfolipids. Together, these findings reveal that T. pseudonana has evolved a sophisticated response to P deficiency involving multiple biochemical strategies that are likely critical to its ability to rapidly respond to variations in environmental P availability. T. pseudonana (Strain 1335 from the Provosoli-Guillard National Center for the Culture of Marine Phytoplankton (CCMP)) was grown in a modified f/2 medium made from Sargasso Sea water. Macronutrients and vitamin B12, biotin, and thiamine solutions were treated with prepared Chelex-100 resin to remove trace metal contaminants followed by trace-metal clean syringe sterilization to yield final nutrient concentrations of 882 μM NaNO3 and 106 μM Na2SiO3, and vitamin concentrations of 75 pM, 400 pM, and 60 nM, respectively. The Fe concentration was also modified from f/2 to 400 nM. All conditions were run in triplicate at 14 ºC, in constant light (120 µmol photons m-2 s-1). Cells were grown with f/2 phosphorus concentrations (P-replete; 36 µM PO4) and with low phosphorus concentrations (P-deficient; 0.4 µM PO4). Growth was monitored daily with cell counts. Duplicate P-replete treatments were pooled and harvested in mid log phase, and triplicate P-deficient treatments were pooled, and also quickly harvested onto 2 µm filters at the onset of P depletion. All RNA samples were snap frozen in liquid nitrogen. Replicate P-deficient cultures were refed to 36 µM phosphate at the onset of P depletion, and subsequently resumed growth. Additional growth studies were performed as described above substituting glycerolphosphate and adenosine monophosphate at 36 µM, for the phosphate in the medium.

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Quantitative contributions of cyanobacterial alkaline phosphatases to biogeochemical rates in the subtropical North Atlantic

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