Mechanisms of increased Trichodesmium fitness under iron and phosphorus co-limitation in the present and future ocean

Nitrogen fixation by cyanobacteria supplies critical bioavailable nitrogen to marine ecosystems worldwide; however, field and lab data have demonstrated it to be limited by iron, phosphorus and/or CO2. To address unknown future interactions among these factors, we grew the nitrogen-fixing cyanobacterium Trichodesmium for 1 year under Fe/P co-limitation following 7 years of both low and high CO2 selection. Fe/P co-limited cell lines demonstrated a complex cellular response including increased growth rates, broad proteome restructuring and cell size reductions relative to steady-state growth limited by either Fe or P alone. Fe/P co-limitation increased abundance of a protein containing a conserved domain previously implicated in cell size regulation, suggesting a similar role in Trichodesmium. Increased CO2 further induced nutrient-limited proteome shifts in widespread core metabolisms. Our results thus suggest that N2-fixing microbes may be significantly impacted by interactions between elevated CO2 and nutrient limitation, with broad implications for global biogeochemical cycles in the future ocean.


Supplementary Figure 2
Protein abundances of Fe-and P-stress proteins in all conditions. The symbols denote statistical significance relative to either the r380 (square) or both the r380 and r750 (triangle). The star denotes proteins only detected in phosphorus limitation. Error bars are standard errors.  Defining the dynamic interplay between these two limitations can be obscuring especially in terms of co-limitation due in part to a strict interpretation of Liebig limitation where one nutrient is primarily limiting and the next most limiting nutrient becomes secondarily limiting rather than co-limiting 1 . However, true co-limitation can be more accurately defined as two or more limiting nutrients simultaneously influencing growth rather than sequentially. Hence, three different definitions of co-limitation have emerged that attempt to elucidate its biochemical underpinnings 2 . Type I aka independent nutrient co-limitation involves two potentially growth limiting nutrients that are generally mutually exclusive in explicit biochemical function. Although most elements are generally interrelated on some level in the cell, this definition pertains to elements that are directly and functionally unrelated and has been used to describe, for example, nitrogen-phosphorus co-limitation. Type II or biochemical substitution colimitation is defined as two elements that can substitute for the same biochemical role within the cell and can transpire in two different manners. The first happens when two elements can substitute effectively within the same enzyme as in zinc-cobalt colimitation 3 , and the second involves two enzymes that can carry out the same biochemical function but each utilizing a different element. Type III, or biochemically dependent colimitation refers to an element becoming limiting as a result of the inability of the cell to sufficiently acquire the other element, as is the case with zinc-phosphorus co-limitation 4 .

Supplementary Note 2
The minimal segregation of P-limited and nutrient-replete proteomes between CO 2 concentrations ( Fig. 1c; Supplementary Fig. 1) indicates that the observed growth rate increases in high CO 2 under replete and P-limitation conditions may either be due to non-protein regulatory controls such as epigenetics 5 , or to other portions of the undetected proteome.
Conversely, since both iron-limited treatments (Fe and Fe/P) had significantly differentiated proteomes based on CO 2 concentration (Fig. 1c, Supplementary Fig. 1), devoid of differences in growth rates between the two CO 2 adaptation regimes (Fig. 1b), the combination of high CO 2 with either Fe-limitation or Fe/P co-limitation seems to induce a broad biochemical response that is flexible enough to maintain growth rates irrespective of CO 2 . This marked yet phenotypically undetected biochemical segregation provides valuable insights into metabolic pathways that may come under biochemical pressure -and their corresponding cellular compensatory mechanisms -when iron is both limiting and co-limiting at future CO 2 levels.

Supplementary Note 3
Since the 380 and 750 μatm CO 2 cell lines were acclimated for ~7 years prior to the experiment, evidence of their adaptive departure from traditional short-term CO 2 physiologies as seen in other studies was shown through reciprocal transfers and diel N 2 fixation measurements 5 . Additionally, nutrient limited cell lines were allowed to acclimate for ~1 year to simultaneously limiting phosphorus and iron concentrations before being subjected to either Fe-or P-single limitation, respectively (See Methods).
This long-term nutrient-limited steady state reflects a significant departure from traditional nutrient limitation experiments where typically, nutrients are either abruptly removed from replete cultures followed by brief acclimation or subsequently removed step-wise until cell death. Conversely, our measurements came after long-term exposure to simultaneous low iron and phosphorus concentrations, which may have altered proteome responses typically seen in short-term limitation studies as previously observed. Intriguingly, these enhanced protein changes in expression patterns were not reflected in differences in growth rates.