Award: OCE-1416673

Using an open ocean/estuarine comparative approach, this project combined oceanographic and advanced molecular techniques to characterize the adaptive responses of microbial communities to multiple stressors using both controlled mesocosm manipulations and natural environmental variability. The project tested the hypothesis that organisms in highly pH-variable environments (coastal) are better adapted to ocean acidification (OA) compared to organisms in more stable environments (open ocean). To test this hypothesis, a novel bioinformatics pipeline was developed and applied to analyze available metagenomic data from coastal and open ocean samples. Consistent with the overarching hypothesis, it was found that the open ocean protein sequences are significantly enriched in polar (basic) amino acids relative to their coastal counterparts, and this pattern does not seem to be a phylogenetic bias of the taxa found in the samples, or attributable to genomic GC% biases or other systematic artifacts. This represents an important finding that reveals one mode of adaptation of microorganisms to the effects of OA. To further test the overarching hypothesis, replicated mesocosms were performed under controlled conditions in the laboratory that examined the effect of both decreasing pH and increasing temperature. In particular, two replicates for each of the following incubations (a) control (b) acidified (-0.3 from the in situ pH) (c) warmed (+3°C from the in situ temperature) (d) warmed and acidified mesocosms were established with either coastal or open ocean water as inoculum. Each mesocosm was maintained under stable conditions for 5 days. Thus, these mesocosm experiments focused on short-term, physiological adaptations to OA with the expectation to observe less gene expression responses from the coastal communities relative to the open ocean communities. Somehow surprisingly, no significantly differentially expressed genes between the control and acidified incubations were detected for either coastal or open ocean water mesocosms. However, the warmed mesocosms showed significantly different gene expression levels for at least 5 functional categories in the coastal incubations (urea subunits and decomposition, ammonia assimilation, DNA repair and serine biosynthesis) and for the majority of identified functional categories (120/160 subsystems) for the open ocean incubations. These results indicate that a relatively small change in pH (e.g., 0.3 units) does not substantially affect microbial community gene expression and activities. In contrast, the temperature treatment elicited much larger gene expression differences in the open ocean relative to the coastal microbial communities, consistent with the hypothesis that open ocean microbial communities may be more vulnerable to climate change. Thus, while warmer waters may enhance overall metabolic rates, there will be long-term shifts in microbial communities and potentially in functions, especially in relatively thermally-stable regions of the (open) oceans. Two manuscripts have been published that described work supported, at least in part, by this project (Tzementzi et al., Nature 2016; Ruiz-Perez et al., Env. Microb. Reports 2019); another two manuscripts are being prepared for publication at the time of this writing. The project has trained one MS student, one PhD student and one postdoctoral research associated at the interface of microbial ecology, ocean biogeochemistry and computational biology (bioinformatics sequence analysis). The metagenomic and metatranscriptomic data produced as part of the project were released under a new BCO-DMO account(project #535573; https://www.bco-dmo.org/award/535573). In summary, these findings advance current understanding of the physiological (gene expression) and molecular biology (amino acid substitutions patterns) of abundant as well as rare members of the natural planktonic oceanic microbial communities to environmental stressors related to OA such as fluctuating pH and temperature. Novel bioinformatic tools and approaches were developed for fine-level resolution analysis of microbial communities and individual populations (e.g., see Tzementzi et al., Nature 2016), which can be applied in other experimental systems or natural habitats. Last Modified: 03/01/2020 Submitted by: Konstantinos T Konstantinidis