Resolving the paradox: Continuous cell-free alkaline phosphatase activity despite high phosphate concentrations
Introduction
Microbes are the engines driving the biogeochemical cycles, underpinning marine food resources and affecting oceanic productivity and climate (Falkowski et al., 2008). It is recognised from the ‘size-reactivity model’ that microbes selectively prefer high molecular weight dissolved organic matter (DOM) due to its superior nutritional quality and bioavailability (Amon and Benner, 1996; Benner and Amon, 2015). A large fraction of this preferred high molecular weight DOM is too big to be transported directly across prokaryotic cell membranes, but rather has to be cleaved outside of the cell or in the periplasmic space by hydrolytic enzymes into low molecular weight DOM (<600 Da) prior to microbial uptake (Weiss et al., 1991). Though, there have been some recent exceptions to this rule for specific polysaccharides (Cuskin et al., 2015; Reintjes et al., 2017), microbial extracellular enzymatic activity (EEA) is recognised as the rate limiting step in the degradation of organic matter in the oceans (Hoppe, 1991), and indispensable for the recycling of limiting nutrients such as phosphate (Pi) from the DOM pool (Duhamel et al., 2010; Arnosti, 2011).
There are two forms of EEA. Cell-associated ectoenzymes are attached to the outside of the microbial cell wall or located in the periplasm while cell-free or dissolved ectoenzymes are completely free of the cell, suspended in the water column and upon release from the cell, remain active for extended periods of time (Hoppe et al., 2002; Baltar, 2018). Until recently, research on marine microbial EEAs has mostly focused on the cell-associated or total EEA, as they were considered to be the only abundant extracellular enzymes in the environment (Hoppe, 1983; Hoppe et al., 2002) and consequently, the only form of ecological significance (Rego et al., 1985; Chróst and Rai, 1993). This cell-associated model for EEAs strongly influenced how these enzymes were assayed, with the cell-free fraction rarely assessed separately.
However, many studies have consistently shown that cell-free extracellular enzymes can make up a significant proportion (often >50% and up to 100%) of the total extracellular enzyme pool (Kamer and Rassoulzadegan, 1995; Li et al., 1998; Baltar et al., 2010; Duhamel et al., 2010; Allison et al., 2012; Baltar et al., 2013; Baltar et al., 2016b; Baltar et al., 2019a). This finding has caused a major conceptual shift in marine EEA research, generating many new questions about the roles of this cell-free fraction, their potential controls, and how they may respond to the pressures of climate change (Arnosti, 2011; Baltar et al., 2016b; Baltar, 2018; Baltar et al., 2019b). These cell-free enzymes can be directly released by microbes in response to the detection of appropriate substrates (Alderkamp et al., 2007), but can also come from several sources in the marine environment including from grazing of protists on bacteria (Hoppe, 1991; Bochdansky et al., 1995), microbial starvation (Chróst, 1991), and in response to stress and/or viral mortality (Kamer and Rassoulzadegan, 1995; Baltar et al., 2019b). Cell-free enzymes have been shown to last from a few days up to several weeks (Li et al., 1998; Ziervogel et al., 2010; Steen and Arnosti, 2011; Baltar et al., 2013). This gives cell-free enzymes the potential to decouple, both temporally and spatially from the producing cell (Arnosti, 2011; Baltar et al., 2016b). This decoupling suggests that the history of the water mass could be more informative in understanding the cell-free enzyme activities than the in situ microbial community at the time of sampling (Kamer and Rassoulzadegan, 1995; Baltar et al., 2010; Arnosti, 2011; Baltar et al., 2016b).
Dissolved inorganic phosphate (Pi) along with inorganic N, plays a key role in the marine environment, being the main limiting macro nutrients for primary production in the open oceans (Björkman and Karl, 1994; Tyrrell, 1999; Björkman and Karl, 2003). Pi is essential for DNA and RNA synthesis as well as energy transfer molecules such as ATP and cell-structures such as phospholipids (Davis and Mahaffey, 2017). Pi is the preferred form of phosphorus for microbial growth as microbes have a high affinity uptake pathway, but Pi is typically limited in the upper ocean (Karl, 2000; Wu et al., 2000; Duhamel et al., 2010). In the euphotic layer of the oceanic water column, the dissolved organic phosphorus (DOP) concentrations are typically at least one order of magnitude higher than those of Pi (Björkman and Karl, 2003; Duhamel et al., 2010; Moore et al., 2013), highlighting the requirement for extracellular alkaline phosphatases (APase) to cleave the required Pi from DOP. The production of extracellular enzymes is a costly energetic process. Thus, in theory extracellular enzymes would only be produced if the preferred Pi is not freely available for uptake. High APase activity is usually explained as a response to Pi limitation (Li et al., 1998; Hoppe, 2003; Duhamel et al., 2010). However, high Pi concentration alongside the high APase has been frequently reported (Chróst, 1991; Koike and Nagata, 1997; Li et al., 1998; Hoppe and Ullrich, 1999; Tamburini et al., 2002; Baltar et al., 2009; Baltar et al., 2016c), contradicting this reasoning. This observed inconsistency between Pi concentrations and APase activities generated the alkaline phosphatase paradox, which has puzzled marine researchers for decades (Chróst, 1991; Hoppe, 2003; Baltar et al., 2009; Baltar et al., 2016c).
This study built on the advances in the understanding of cell-free versus cell-associated EEAs in the last decade to find an explanation for this long-time paradox. Coastal and open-ocean (epi- and mesopelagic waters) waters were repeatedly sampled (biweekly for 1 year the coastal sampling and bimonthly for 9 months the open ocean sampling), and lab experiments were performed to study the dynamics of APase (cell-free and cell-associated) activity and Pi concentration. It was hypothesised that the paradox can be explained by an uncoupling between APase and Pi provoked by a continuous high proportion of cell-free APase in marine environments. This is based on previous findings reporting that cell-free APase makes up a substantial proportion of the total APase pool, and that these cell-free APase can have a relatively long residence time in the environment.
Section snippets
Study sites and sampling
A biweekly coastal sampling was carried out at the University of Otago's Portobello Marine Laboratory, situated on the Otago Harbour, Dunedin, New Zealand for over 1 year, between August 2017 and September 2018. Otago Harbour is a tidal inlet, consisting of two distinct basins (Heath, 1975; Grove and Probert, 1999). The laboratory is based at the outer basin, which has waters similar in composition to coastal seawater with short residence times in exchanging with the open sea (Rainer, 1981;
Results and discussion
In the Portobello coastal waters, APase activity was detected throughout the year despite pronounced seasonal changes in temperature (6.91–21.70 °C) (Fig. 1a) and Pi concentrations (0.13–0.83 μM) (Fig. 1b). This total APase activity ranged between 8.45–39.07 nmol l−1 h−1 (Fig. 1b). The cell-free fraction represented the majority of total APase throughout the study (ranging from 71.5–99.4%) demonstrating the relative dominance of cell-free APase. Overall, there was not a clear relationship
Acknowledgments
We would like to thank the skippers and crew of the RV Polaris II for their help during the sampling events, and the technical support by Doug Mackie, Linda Groenewegen and Reuben Pooley at the Portobello marine lab. This research was supported by a University of Otago research grant and a Rutherford Discovery Fellowship (Royal Society of New Zealand) to FB. GJH was supported by the Austrian Science Fund (FWF) project ARTEMIX (P28781-B21).
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