the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 04 Mar 2025
Part 2: 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 often studied through enzyme rate-based estimates and nutrient-amendment bioassays, but these approaches are limited in their 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.
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RC1: 'Comment on egusphere-2024-3996', Anonymous Referee #1, 08 Apr 2025
General:
The manuscript, No.: egusphere-2024-3996, Noelle A. Held et al., “Part 2: Quantitative contributions of cyanobacterial alkaline phosphatases to biogeochemical rates in the subtropical North Atlantic,” integrates multiple methods to address questions about alkaline phosphatases in marine cyanobacteria and how these enzymes respond to nutrient limitation and different trace metals. The experiments and results appropriately address the questions posed by the authors about the abundance of these enzymes under different biochemical conditions and exposure to different metal cofactors. The experiments also revealed the unexpended finding of potentially promiscuous cofactor binding that remain open for future studies. The conclusions regarding biological complexity, the discussion of methodological caveats, and suggested future directions are appropriate for the outcomes of this current study. Overall, the design of the study, the method application of absolute quantitative proteomics, and the potential interest for the oceanographic and proteomics communities is good.
My main concerns after review are regarding the structure of the results section (details below) and whether this Part 2 manuscript is meant to be more of a methodological highlight, complementary to the in situ nutrient data and future climate discussion points in Part 1. Or, if the focus of both manuscripts is ultimately on the environmentally contextualized results (e.g., Figure 3). I would like the authors to comment on the specific comments/questions listed below:
Specific comments:
If this Part 2 manuscript is meant to be an example application of “absolute quantitative proteomics” as a method, then it would benefit from a schematic methods comparison of the alkaline phosphatase rates in the “traditional” bioassay (Figure 1) vs. the strain-resolved approach (Figure 2).
The alkaline phosphatase activity rates in the Figure 1 legend are also not clearly explained in the methods section.
- If the "significant responses" portion of the methods is the section that covers these rate calculations just spell that out a bit more.
- If the rate calculations used in the "second to left column" in Figure 1 are based on the same principles used in the metalloproteome section that comes later regarding the absolute quantitative measurements then this should be made more clear. Either move the equations 1a - 2b to the methods and link them also to Figure 1 in the text or more clearly explain the rate calculations that went into Figure 1.
Results) Add a map of sampling stations to the “Biogeochemical setting” section. This would help orient the reader in the beginning of the Results section to understand the stations, environmental concentrations, and to which samples/stations the following assay results (Fig. 1 & 2) belong.
- This could be done by either adding a simple map of the sampling stations in addition to Table 3/Figure 1 or moving the section with the map (Figure 3 and associated text) up to the start of the Results section.
- Consider if the story works with Figure 3 and the APA discussion first and if not just add a simple station map before Figure 1.
Figure 1) The main purpose of Figure 1 is to show that there were no significant differences in any of the “conventional parameters” measured at all four stations (i.e., a point of comparison for the significant differences in the next method presented) - this plot should be either condensed or simplified.
- A condensed Figure 1 should show only the Alkaline Phosphatase rate column at the four different stations, considering this is the more important comparison point for the next analysis in Figure 2 (leaving the other conventional parameters that are also not statistically significantly changing, Chl a and cell counts, to the text and supplement).
- If the bar plots are all kept together, simplify this plot by using a letter system (a – d) for the different parameters and incorporate this into the figure legend to make it easier to navigate than “left column” vs. “second to left column.”
348) Are there any citations from model organism studies where they found multiple isoforms in the same organism?
371) Not really an accurate use of “co-evolution," as this is a biological term typically defined by two species influencing each other's evolution. Be cautious with referring to chemical evolution and the biological evolution of a specific lineage of organisms in the same sentence. Maybe “the evolution of cyanobacteria in the dynamic chemical conditions of the ancient ocean…”
430) Is there a citation for phosphorus stress studies? Unless it is covered with the Saito 2011b in the next sentence.
For the PRIDE data submission, add a sample description .csv file to the uploaded data that provides the MS file names and the corresponding stations and incubation conditions to help others navigate the data download for potential future reanalysis.
Technical corrections:
275) add parentheses around the i.e. statement to match line 274
308) provide the station coordinates or station name in parentheses after “westernmost”
374) Watch out for changing between chemical symbols and full names. So far it has been very consistent, this was the only instance I spotted a full name.
411-417) Figure 3 legend: Citations are doubled.
420 – 424) Same citations having the doubling issue.
453) Supplement table referenced here should be Tables S3 and S4
In general check that the references to the supplement tables/figures are correct. Some of them seem to be switched around and supplement tables S5 -S7 and Figures S1-S5 are not referenced in the text. Last supplement figure should also be Figure S6.
Citation: https://doi.org/10.5194/egusphere-2024-3996-RC1 -
RC2: 'Comment on egusphere-2024-3996', Anonymous Referee #2, 06 Jul 2025
Held and others study cyanobacterial alkaline phosphatases in the North Atlantic. The manuscript was well-written and well-referenced, and I only have a few minor comments to improve accuracy and readability.
The abstract would benefit from numerical values
Minor spacing errors, especially adjacent to references; note erroneous text on 125, double-reference on 420, etc. The manuscript needs to be carefully re-read for accuracy.
193: lack of reference concerning but if there are no existing references this should be justified in more detail
225: let the reader decide if the approach is accepted; it merely has been used before
Discussion points enter the results section, like in 303 ‘represents an interesting avenue for future research’. Is the results meant to be results and discussion? It certainly reads this way.
The requirement for the reader to distinguish between red and green in Figure 3 needs to be reconsidered.
449: ‘the PhoX isoform’
460 and possibly elsewhere: don’t use the * symbol to represent multiplication in formal mathematical equations, use the multiplication symbol.
497: 0.0347 nM h-1 is remarkably specific.
Citation: https://doi.org/10.5194/egusphere-2024-3996-RC2
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