Microscopic algae that live in the ocean release countless tons of oxygen into the atmosphere each year. Widespread algae – known as coccolithophores – surround their little plant-like body with a mineral shell made of a material similar to chalk. These microscopic algae form seasonal blooms. Over several weeks in early summer, the algae grow to enormous numbers and cover hundreds of thousands of square kilometers in the ocean. These blooms become so vast that satellites can detect them. However, suddenly the blooms collapse; the algae die and their chalky shells sink to the bottom of the ocean where they have been accumulating for millions of years.

More and more evidence suggests that these tiny algae interact with bacteria in various ways. However, so far, no one had documented a direct interaction between bacteria and a member of this key group of algae.

Now, in a controlled laboratory environment, Segev et al. show that marine bacteria from the Roseobacter group physically attach onto a tiny coccolithophore alga called Emiliania huxleyi. While the bacteria are attached to their algal host, they enjoy a supply of nutrients that trickles from the algal cell. Unexpectedly, Segev et al. also discovered that the algae grow better in the presence of the bacteria. It turns out that the bacteria use a molecule that they obtain from their algal hosts to produce a small hormone-like molecule that in turn enhances the growth of the algae. However, after three weeks of growing together, the bacteria produce so much of the growth-enhancing molecule – which is harmful in higher concentrations – that they actually kill their algal host.

These findings suggest that the bacteria first promote the alga’s growth to boost their supply of nutrients. But as algae grow older, the bacteria harvest the algae to enjoy a last pulse of nutrients and allow their offspring to swim away and attach to younger algae.

The next challenge will be to link these laboratory observations to the actual microbial interactions in the ocean. It will also be important to explore whether other algae and bacteria interact in similar ways and if bacteria contribute to the sudden collapse of algal blooms by killing the algae.

There are many microbes that have influenced Earth's biogeochemistry. Prime among these are the coccolithophores, a diverse group of unicellular marine algae of the haptophyte division. Because of their high abundance, these micro-algae are fundamental in the global oxygen, carbon, and sulfur cycles (Balch et al., 1991; Beaufort et al., 2011; Simó, 2001; Field et al., 1998). As a consequence of their photosynthetic capacity these algae, together with other phytoplankton, are responsible for nearly half of our planet’s primary production (Field et al., 1998). Coccolithophore cells are usually surrounded by elaborate platelets made of crystalline calcium carbonate (calcite) referred to as coccoliths. During coccolith production, carbon dioxide is released and can escape from the ocean to the atmosphere (Marsh, 2003). More importantly, coccoliths serve as a carbon sink as they accumulate on the bottom of the oceans (Sabine et al., 2004). Coccolith production by E. huxleyi accounts for roughly 1/3 of the total marine calcium carbonate production (Iglesias-Rodríguez et al., 2002). Hence, coccolithophores play a complex role in the global carbon cycle.

Emiliania huxleyi is the most widespread coccolithophore in modern oceans, forming dense annual blooms (Paasche, 2001). The blooms can cover thousands of square kilometers of ocean surfaces and are easily detected by satellites due to the highly reflective nature of the coccoliths (Balch et al., 1991; Holligan et al., 1983). The blooms also exhibit unique dynamics; they form seasonally over several weeks and then suddenly collapse (Behrenfeld and Boss, 2014; Lehahn et al., 2014; Tyrrell and Merico, 2004), a process that has been attributed to viral infection (Bratbak et al., 1993; Lehahn et al., 2014; Vardi et al., 2012). Recent evidence suggests that environmental stresses and viral infection can trigger oxidative stress and a process similar to programmed cell death (PCD) in E. huxleyi (Bidle et al., 2007; Vardi et al., 2009; Bidle, 2016). The induction of PCD, which is an autocatalytic process, has been shown to occur in various widespread species of phytoplankton including E. huxleyi, and functional links have been demonstrated between viral infection, PCD, and algal bloom collapse (Bidle, 2015, 2016; Bidle and Vardi, 2011; Fulton et al., 2014; Vardi et al., 2009, 2012; Rohwer and Thurber, 2009). Interestingly, although E. huxleyi blooms harbor a rich community of bacteria, at times dominated by the Roseobacter group (González et al., 2000; Green et al., 2015), bacteria are not generally considered to be a factor influencing coccolithophore physiology and bloom dynamics.

Various types of phytoplankton were shown to have both mutualistic and antagonistic interactions with bacteria (Amin et al., 2015; Miller and Belas, 2004; Miller et al., 2004; Wang et al., 2014; Durham et al., 2015). In addition, the possible role of algicidal bacteria in the ocean has been examined and discussed (Mayali and Azam, 2004; Harvey et al., 2016). It has been previously suggested by our laboratories that bacteria might interact with E. huxleyi (Seyedsayamdost et al., 2011). However, coccolithophore-bacteria interactions have not yet been unambiguously demonstrated. This gap is curious because E. huxleyi's important role in the global sulfur cycle is in part a consequence of an algal-bacterial interaction. E. huxleyi produces the osmolyte and antioxidant dimethylsulfoniopropionate (DMSP) (Sunda et al., 2002). This molecule, when released into the water by leakage or cell lysis, can be used by some bacteria as a source of sulfur and carbon (Curson et al., 2011; González et al., 1999). During DMSP catabolism, bacteria such as Roseobacters produce the volatile by-product dimethyl sulfide (DMS). E. huxleyi is also a producer of DMS, which is a bioactive gas with possible roles in climate regulation (Charlson et al., 1987; Alcolombri et al., 2015).When DMS enters the atmosphere it is oxidized and serves to form cloud condensation nuclei (Curson et al., 2011; González et al., 1999). While the DMSP flux from algae to bacteria, and the production of DMS gas by both algae and bacteria have been clearly demonstrated, the role of DMS in climate regulation has been questioned (Quinn and Bates, 2011).

Accumulating evidence suggests that there may be widespread interactions between E. huxleyi and Roseobacters. Phaeobacter inhibens (Buddruhs et al., 2013), a well-studied member of the Roseobacter group, was shown to produce molecules that specifically affect E. huxleyi (Seyedsayamdost et al., 2011). This bacterium, when grown in a pure culture in the presence of p-coumaric acid, a product released by aging algae, produced novel compounds able to lyse E. huxleyi. The compounds were named roseobacticides and their discovery pointed towards a possible interaction between P. inhibens and E. huxleyi (Seyedsayamdost et al., 2011). Furthermore, we recently showed that lipid metabolism in E. huxleyi is altered in the presence of P. inhibens (Segev et al., 2016). However, a direct physical interaction between these algae and bacteria had not been previously described and no other details of their interaction were known. Here we describe the establishment of a co-culture model system between E. huxleyi and P. inhibens that allows the examination of the spatiotemporal dynamics of their interaction. We provide evidence that E. huxleyi and P. inhibens associate intimately when co-cultured. We show that bacteria promote algal growth but eventually kill their aging algal hosts. The same bacterial compound, indole-3-acetic acid, mediates stimulation of algal growth as well as algal death. Finally, algal death in the co-culture seems to involve an apoptotic process. Similar E. huxleyi - bacteria interactions might occur in the ocean and could thus affect algal physiology, bloom dynamics and biogeochemical cycles.

E. huxleyi and P. inhibens are two well-studied marine microbes. To determine if they co-occur in algal blooms we analyzed the bacterial community associated with E. huxleyi blooms using a culture-independent metagenomic approach. Two independent blooms were sampled in the Gulf of Maine during the summer of 2015. The results shown in Figure 1 indicate that P. inhibens was indeed found co-occurring with E. huxleyi in algal blooms (Figure 1). Thus, the suggested interaction between these microorganisms might be ecologically significant. To study the interactions of E. huxleyi and P. inhibens, it was necessary to establish conditions to co-culture these two species. We started by examining pure cultures of each microorganism. Coccolith-forming (i.e. calcifying) E. huxleyi (strain CCMP3266) were inoculated into L1-Si, a seawater based medium supplemented with additional sources of phosphorus (0.04 mM PO₄), nitrogen (0.9 mM NO₃) and sulfur (0.08 µM SO₄), along with vitamins and trace metals (Guillard and Hargraves, 1993) (see Materials and methods). In this medium, E. huxleyi grows to 3×10⁵ cell/ml. Under these conditions E. huxleyi produces calcium carbonate coccoliths that surround the algal cell (Figure 2a). P. inhibens DSM17395 is normally grown in the rich medium 1/2YTSS (Seyedsayamdost et al., 2011) (see Materials and methods) where it easily aggregates; it often forms ‘rosette’ structures through a polysaccharide-containing pole (Figure 2b,c) (Segev et al., 2015). Of note, alone these bacteria do not grow in the L1-Si medium (Figure 2d, grey bars). However, we found that bacteria do grow in co-culture with E. huxleyi. To grow a co-culture, we inoculated algae into L1-Si medium and, after four days, introduced bacteria into the algal culture. In these co-cultures, bacterial numbers increased nearly five orders of magnitude over a period of 14 days (Figure 2d, green bars). Microscopic examination of the co-culture revealed that some algae were no longer surrounded by coccoliths (Figure 2e). Rather, naked algal cells were now covered by bacteria attached via their poles. This attachment was evident in both fixed (Figure 2e) and live (Figure 2f) samples. Of note, attachment of P. inhibens to other micro-algae as well as macro-algae has been previously demonstrated (Frank et al., 2015). Using a specific fluorescent lectin to detect the polar polysaccharide (see Materials and methods), it appeared that bacterial attachment onto the algal cell also involves the polar bacterial polysaccharide (Figure 2f). Examination of co-cultures revealed that over time more algae have attached bacteria (Figure 2g) and each algal cell is associated with increasing numbers of bacteria as the co-culture ages (Figure 2f,h).

Figure 1

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Figure 2

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Bacteria clearly benefit from interacting with the algal host as their growth is enabled by the algae in an otherwise non-permissive medium (Figure 2d). What do bacteria receive from algae that allows them to grow? Given that L1-Si does not contain significant amounts of organic carbon to permit robust growth of the heterotrophic bacteria, it stands to reason that the key nutrient that algae provide is fixed carbon. If indeed fixed carbon were to be the sole nutrient needed by the bacteria, addition of a utilizable form of carbon to L1-Si should enable bacterial growth. However, addition of 5.5 mM glucose did not lead to significant bacterial growth (Figure 3a). This was an unexpected result because, as mentioned above, L1-Si in addition to seawater also contains added phosphorus (0.04 mM PO₄), nitrogen (0.9 mM NO₃) and sulfur (0.08 μM SO₄). In fact, even addition of higher nutrient concentrations in forms shown to be utilizable by P. inhibens (Zech et al., 2009) as individual supplements (nitrogen 5 mM NH₄, phosphorous 2 mM PO₄, and sulfur 33 mM SO₄) or in various combinations of two or three of them did not lead to robust bacterial growth (Figure 3a). Only addition of all four essential nutrients resulted in bacterial growth to a density of 5×10⁸ CFU/ml, which we normalized to 100% in Figure 3a. Thus, E. huxleyi can provide all four essential nutrients (C, N, P and S) in suitable forms and concentrations to enable growth of the heterotrophic bacterium P. inhibens.

Figure 3

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The sulfur flux from algae to bacteria is of special interest. Because of its ecological importance, we wanted to investigate whether DMSP plays a role in the interaction between E. huxleyi and P. inhibens. First, we examined the ability of bacteria to grow with DMSP as a sole source of sulfur or carbon. As shown in Figure 3a, DMSP can serve as a sulfur source (Figure 3a, 'CNP + DMSP 30 μM'). In contrast, DMSP does not supply sufficient carbon to support robust bacterial growth. (Figure 3a 'NP + DMSP 30 μM'). Even when DMSP was added in higher concentrations to supply carbon in a comparable amount to the carbon supplied by the 5.5 mM glucose in the parallel experiments, bacterial growth was not evident (Figure 3a ‘NP + DMSP 6mM’). Next, we directly monitored DMSP consumption in a growing bacterial culture. Our measurements indicate that all of the added DMSP is rapidly utilized by the growing bacteria whereas in un-inoculated medium the DMSP levels remain relatively stable over time (Figure 3b). Based on these observations, we then proceeded to determine whether DMSP is produced and exuded by algae in pure culture and in co-cultures. Indeed, over a period of 17 days, DMSP concentration in the medium of a pure algal culture increased, reaching a concentration of nearly 6 µM (Figure 3c, black bars). In contrast, in a co-culture of algae and bacteria, DMSP levels in the medium were nearly undetectable, presumably due to its rapid metabolism by the bacteria (Figure 3c, grey bars). Since bacteria are directly attached to their algal host it is possible that they experience a considerably higher local concentration of DMSP than the concentration measured in the bulk medium. It has been reported that Roseobacters and other bacteria can chemotax towards DMSP and catabolize it and various metabolic pathways for the bacterial use of the DMSP sulfur have been proposed and tracked (Miller and Belas, 2004; Miller et al., 2004; Seymour et al., 2010; Brock et al., 2013; Wang et al., 2016). Thus, it is possible that DMSP serves as a chemical cue attracting bacteria to colonize the E. huxleyi host cell. Additional experiments are clearly needed to determine if indeed DMSP serves as an infochemical promoting bacterial colonization. Yet, it has been previously shown that algal DMSP and exudates serve as a strong cue to attract bacteria (Seymour et al., 2010; Smriga et al., 2016).

To further characterize the algal-bacterial interaction we explored the bacterial effect on algal growth. Flow cytometry is commonly used to monitor algal growth. However, due to bacterial attachment and algal clumping, our co-cultures consist of aggregates of cells of varying sizes. Thus, it is challenging to accurately interpret the results of flow cytometry analyses. Therefore, our analyses included traditional flow cytometry as well as validation of our results using imaging cytometry in order to characterize the different particles (see Materials and methods). We found that during the initial 10 days of culturing, there were greater numbers of algae in the co-cultures compared to pure algal cultures (Figure 4a). In the co-culture, algae reach approximately 25% higher numbers in comparison with the maximum reached in the absence of bacteria. In addition, in an algal pure culture the death phase starts after day seven while in a co-culture a significant decrease in population is evident only at day 17. Interestingly, the death phase in an algal pure culture seems more gradual than the rapid demise observed in a co-culture (Figure 4a). Thus, it seems that the algal-bacterial interaction is dynamic. Initially the interaction is mutualistic, however over time bacteria may become harmful for their algal hosts.

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Various bacteria that interact with plants are known to provide phytohormones (auxins) that promote plant growth (Costacurta and Vanderleyden, 1995). It was previously suggested that phenylacetic acid (PAA) produced by P. inhibens might serve as an auxin to enhance algal growth (Seyedsayamdost et al., 2011). We could not detect PAA in bacterial cultures or algal-bacterial co-cultures.

Intriguingly, the auxin indole-3-acetic acid (IAA) was previously demonstrated to play significant roles in numerous terrestrial plant-bacteria interactions and was recently shown to be key in a marine association between a diatom and a Roseobacter bacterium (Amin et al., 2015; Spaepen et al., 2007). As many members of the Roseobacter group have multiple metabolic pathways for IAA synthesis (Moran et al., 2007), we posited that IAA might be produced by P. inhibens to promote the growth of E. huxleyi in co-culture. To test this, we examined whether IAA is produced by P. inhibens. We were able to detect IAA (0.4 nM) in bacterial pure cultures (see Materials and methods) (Figure 4b). A standard of IAA was analyzed by high resolution LC-MS and targeted MS/MS (see Materials and methods). The retention time (Figure 4b), exact mass (Figure 4—figure supplement 1a), and fragmentation pattern (Figure 4—figure supplement 1b) of the standard matched with the IAA that was detected in bacterial cultures. Importantly, no IAA was detected in axenic algal cultures. Next we assessed algal growth upon addition of IAA. Similar to the increase we observed in co-culture, algal growth yield was improved by 20% upon addition of 1 µM and 100 µM IAA (Figure 4c).

Our attempts to monitor IAA levels in co-cultures revealed that in these conditions IAA was undetectable, suggesting rapid up-take by algae. This observation is in agreement with previous reports of significant binding of IAA and rapid signal transduction in plants, indicative of the high affinity towards this compound (Kepinski and Leyser, 2005). Interestingly, studies have described microbial signaling pathways taking place in the rhizosphere (the soil immediately surrounding the roots of terrestrial plants) (Spaepen et al., 2007). It is tempting to hypothesize that similar short-circuit processes could take place in the phycosphere (the immediate volume in proximity to the algal cell). Thus, it seems that in our experimental system bacteria inhabiting the algal phycosphere supply their algal host with growth promoting molecules.

To further characterize bacterial IAA production in the algal phycosphere, we examined whether similarly to other IAA-producing bacteria, P. inhibens will alter IAA production in response to exogenous tryptophan (Brandl and Lindow, 1996; Patten and Glick, 2002; Prinsen, 1993; Theunis et al., 2004; Zimmer et al., 1998). Indeed, addition of 0.1 mM tryptophan resulted in the production of 10.8 nM IAA, approximately 25-fold increase in comparison to conditions with no added tryptophan (Figure 5a). The increase in IAA production could be the result of two different mechanisms. Added tryptophan could enhance bacterial metabolism, thus resulting in general increase of bacterially-produced metabolites. Alternatively, exogenous tryptophan could be shunted primarily towards IAA production. To distinguish between these two possibilities, we supplemented a bacterial culture with uniformly labeled tryptophan (¹³C and ¹⁵N). If the labeled tryptophan were taken up and directly shuttled towards IAA production, all IAA should be fully labeled (m/z 187) (Figure 5b). However, if the imported tryptophan participates in other cellular processes, and atoms are exchanged with endogenous pools of carbon and nitrogen, then the resulting IAA will not be fully labeled, thus resulting in a lower mass. The lowest possible mass would be of a fully unlabeled IAA molecule (m/z 176). The results of our experiments indicate that all produced IAA is fully labeled (Figure 5b), thus indicating that exogenous tryptophan is directly converted to IAA. Of note, a recent study reported on the production of IAA in the mM range in axenic E. huxleyi cultures supplemented with tryptophan (Labeeuw et al., 2016). As all of our experiments and controls indicated no IAA production by algae, currently we do not understand the source of this apparent contradiction.

Figure 5

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To elucidate the relevance of increased IAA production by bacteria in the presence of exogenous tryptophan, we wanted to examine whether E. huxleyi exudes tryptophan. If indeed tryptophan is exuded by algal cells, bacteria in the phycosphere could import it and convert it to IAA. Secreted bacterial IAA could be utilized by algae and lead to improved algal yields. Such chemical cross talk will feed into a positive algal-bacterial feedback loop. In line with this idea, in another algal-bacterial interaction it has been shown that tryptophan released by a diatom fuels IAA production by a bacterium (Amin et al., 2015). To monitor bioavailable tryptophan in E. huxleyi exudates, we used an Escherichia coli strain that is a strict tryptophan auxotroph. This bacterial strain relies solely on exogenous tryptophan; in the presence of extracellular tryptophan the strain will grow and in the absence of tryptophan it will not grow. Cultivation of this strain in minimal medium supplemented with filtered algal supernatant resulted in marked bacterial growth (Figure 5c). This indicates that algal exudates contain bioavailable tryptophan. Interestingly, we detected tryptophan (100 nM) in filtered samples of E. huxleyi blooms (see Materials and methods), suggesting that this metabolite may be relevant to environmental interactions.

To better understand the temporal dynamics of the algal-bacterial interaction we examined co-cultures over a period of 17 days and compared them with algal pure cultures of the same age. After 10 days of incubation, no visible difference was apparent between algal pure cultures and co-cultures (Figure 6a,b). However, after 17 days of incubation the color of the co-cultures rapidly changed from green (the color of healthy algae) to white, a process we refer to as ‘bleaching’ (Figure 6c,d and Video 1). To better understand these algal changes we examined the various cultures under the microscope. This revealed that the algal cells in a bleached co-culture exhibit more coccolith shedding (Figure 6e,f,g,h, note arrow in Figure 6h). Furthermore, the typical fluorescent crescent shape that is made of the two chloroplasts in each cell (Figure 6i,j,k) is lost in the bleached co-culture and the fluorescent signal emanating from chlorophyll and accessory pigments fills the entire cell (Figure 6l). In addition, using a viability stain (Sytox green, see Materials and methods) it is evident that the aging co-culture contains mostly dead algal cells (Figure 6m,n,o,p). One possible explanation for the bleaching observed is that the bacteria simply degrade dying algal cells at day 17 while similarly dying algal cells in the pure culture at the same time point remain intact. However, comparison of the death rates in the algal population in pure and co-cultures reveals a significant difference. At this time point, the vast majority of algal cells in the co-culture are dead (94% as indicated by Sytox staining, see Figure 3p) while in the pure culture only 21% are dead by day 17. Thus, the presence of bacteria seems to play a key role in promoting algal death. Taken together, these results suggest that following the mutualistic phase in the algal-bacterial interaction, bacteria become pathogens that cause the bleaching and death of their algal partners.

Figure 6

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Video 1

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To further explore the death process experienced by algae in co-culture, we examined the expression profile of a select group of algal genes. Out of 50 examined genes representing various functional groups (Supplementary file 1), 15 genes exhibited significant up-regulation in algae over time in co-culture (Table 1). To test whether this group of 15 genes exhibits expression levels that are significantly higher than the other 35 genes (in Supplementary file 1), we first calculated the variance in the two datasets at day 17 using an F-test. The variance was not found to be statistically different and thus a two-tailed Student’s T-test was performed assuming two samples with equal variance. The resulting probability of the T-test was 2.8×10⁻¹¹ indicating that the difference between the two datasets is statistically significant. The highly up-regulated genes encode proteins that are presumed to be involved in various aspects of oxidative stress and programmed cell death (PCD). PCD naturally occurs in an aging algal population and can be triggered by a variety of biotic stresses such as viral infection and abiotic environmental stresses such as nutrient limitation and various light regimes (Bidle, 2015; 2016). Our expression data suggest that PCD as well as responses to oxidative stress are more prevalent among algal cells in co-culture. Thus, PCD in E. huxleyi seems to be triggered by bacteria. Of note, the up-regulation of genes encoding proteins that are directly involved in the metabolism of reactive oxygen species (ROS), such as glutathione (Table 1), was previously shown to be essential in viral infection of E. huxleyi (Sheyn et al., 2016). To further investigate if algae in co-culture experience a bacterially-induced process similar to PCD, algal cells from axenic cultures and co-cultures were tested for the presence of degraded DNA, a physiological hallmarks of PCD (Gavrieli et al., 1992). The fluorescent Terminal deoxynucleotidyl Transferase dUTP nick end-labeling assay (TUNEL) was previously established as a reliable method for in situ identification of DNA fragmentation as a PCD indicator in various phytoplankton species (Johnson et al., 2014; Segovia et al., 2003; Berman-Frank et al., 2004). Using the TUNEL assay (see Materials and methods) we could detect that 90% of algal cells in 20-day old co-cultures contain highly fragmented DNA in comparison to 35% in axenic algal cultures of the same age (Figure 6q–t).

To better understand how bacteria cause algal death in our model system, we explored the possible involvement of known algicidal compounds produced by P. inhibens. A previous study carried out in our laboratories reported on a novel group of compounds – roseobacticides – that are produced by P. inhibens and lyse E. huxleyi (Seyedsayamdost et al., 2011). We conducted several experiments to examine whether roseobacticides are the killing agents of algae in aging co-cultures. We successfully reproduced the findings of the previous study (Figure 7). However, our results indicate that while roseobacticides from P. inhibens indeed kill the non-calcifying algal strain CCMP372 used in the previous study, they do not kill the calcifying algal strain CCMP3266 used in the current study (Figure 7a). Importantly, in CCMP3266 cultures no bleaching was seen upon addition of roseobacticides. Moreover, there is no detectable production of roseobacticides in our co-culture conditions (Figure 7b). Thus, roseobacticides do not appear to play a role in algal death in the co-culture experimental system described here.

Figure 7

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One small molecule released by bacteria that does appear to have a role in algal death is IAA. At concentrations between 1 and 100 µM IAA had a positive effect on algal growth, however 1000 µM proved to be harmful to algae (Figure 4c). The hormetic effect of IAA has been previously described; IAA promotes plant growth at low concentration while acting as a growth inhibitor at high concentrations (Persello-Cartieaux et al., 2001, 2003; Xie et al., 1996). Similar effects were observed when increasing concentrations of IAA were added to a culture of diatoms (Amin et al., 2015). In addition, there are morphological similarities between algae that were killed by bacteria in co-culture and algae that were killed by high concentrations of IAA in pure culture (Figure 4d). These observations drove us to hypothesize that IAA can mediate both mutualistic and pathogenic interactions between bacteria and algae. Initially, bacterially-produced IAA promotes algal growth while later on it might be the driving force underlying algal death.

In various bacteria, attempts to perturb IAA synthesis were unsuccessful due to multiple redundant biosynthetic pathways (Spaepen et al., 2007). We examined several mutants of P. inhibens that carry mutations in various pathways of IAA synthesis (Table 2 and Figure 8a). In agreement with previous reports, these mutants were still capable of producing IAA (Table 2) (Spaepen et al., 2007) and cause algal bleaching. Of note, a mutant in tryptophan biosynthesis (Table 2, transposon mutant 1630) was unable to grow in pure culture without addition of tryptophan (data not shown) but exhibited robust growth in co-culture with algae (Figure 8b) and was able to drive algal bleaching. These results further corroborate the presence of tryptophan in algal exudates.

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To test whether high concentration of bacterially-produced IAA could cause algal death, we wanted to promote the biosynthesis of IAA by bacteria in co-culture. Because P. inhibens exhibited a 25-fold increase in yields of IAA upon utilization of exogenous tryptophan (Figure 5a and Table 2), we reasoned that an increase in IAA production should occur when a co-culture is supplemented with exogenous tryptophan. Our results indicate, that a co-culture that has been supplemented with 0.1 mM tryptophan undergoes accelerated death with bleaching occurring one week earlier than in non-treated co-cultures (Figure 9a). Moreover, examination under the microscope of co-cultures that have been treated with tryptophan revealed a remarkable change in bacterial behavior. In cultures supplemented with tryptophan, bacteria became hyper-colonizers and attached to algal cells in significantly higher rates (Figure 9b). Taken together, our observations indicate that tryptophan serves both as a precursor for IAA biosynthesis and as a cue capable of altering bacterial behavior towards their algal host.

Figure 9

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The process in which algal biomass gradually increases to form a bloom, covering vast areas and then abruptly collapses, has attracted much attention from various disciplines. It is known that viral infection is key in the blooms’ demise (Bratbak et al., 1993; Lehahn et al., 2014; Vardi et al., 2012). In our model system the presence of one species of bacteria resulted in the sharp demise of the algal population. While further studies are required in order to explore E. huxleyi-bacterial interactions in the ocean, it is possible that bacterial influences act in concert with viruses to drive the termination of the blooms. Environmental stresses such as nutrient depletion as well as viral infection have been shown to enhance PCD in E. huxleyi (Bidle, 2015, 2016). Similarly, we have observed bacterially-induced PCD. While attached bacteria seem to be trapped on their dying host (Figure 10), their offspring get access to the nutrients from lysed algae and can then swim away and colonize a younger algal cell. Recently, Smirga and colleagues demonstrated experimentally how planktonic bacteria crowd around a lysing microalga, feeding off the released cellular content (Smriga et al., 2016). Our discovery of chemical crosstalk in the phycosphere and the bacterially-mediated algal death were made possible due to the co-culture experimental system that enables investigation over time.

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In the current study tryptophan was identified as a central compound in the E. huxleyi – P. inhibens interaction. This led us to explore its abundance in E. huxleyi natural blooms, where it indeed was found. Previously, tryptophan was identified as a key metabolite in the interaction between a diatom and a Roseobacter (Amin et al., 2015). The use of both targeted and untargeted metabolomics in the lab can greatly aid in identifying metabolites with ecological relevance (Fiore et al., 2015; Johnson et al., 2016; Durham et al., 2015; Amin et al., 2015). As revealed in the current study, when microorganisms are in close proximity, molecules can be produced and rapidly consumed and thus remain undetectable. Local and short-lived signals that are significant cues in the phycosphere would be undetectable in the environment but can be deciphered using model systems similar to the one we have described.

In our study, IAA was identified as a key component of the algal-bacterial chemical crosstalk. The bacterially-produced IAA initially increases algal yields. This growth enhancement is followed by an inevitable death. Clearly IAA exhibits a hormetic effect - it is beneficial in low concentrations and becomes harmful at higher doses. The hormetic nature of IAA has been previously demonstrated in various plant-bacteria interactions (Persello-Cartieaux et al., 2001, 2003; Xie et al., 1996). In the association of the plant pathogen Agrobacterium tumefaciens with its hosts, IAA is central too (Spaepen et al., 2007; Subramoni et al., 2014). It is produced to enhance growth of plant tissue while creating tumors. Several similarities between A. tumefaciens and P. inhibens seem to exist; both attach through their pole to their host (Heindl et al., 2014; Xu et al., 2013), both express a polar polysaccharide involved in bacterial attachment (Heindl et al., 2014; Xu et al., 2013) and both utilize IAA to manipulate the growth of their host (Subramoni et al., 2014). In light of these resemblances, it is possible that additional aspects of the E. huxleyi – P. inhibens interaction will be similar to the mechanisms employed by A. tumefaciens and its interactions with host plants.

We have shown that IAA produced by bacteria enhances algal growth. How is algal growth promoted? One possibility is that algal growth augmentation is the result of changes in light harvesting efficiency. As reported by Falkowski and colleagues, changes in the capability of transducing light energy to chemical energy and eventually to biomass will result in changes in growth (Falkowski et al., 1985). Since all our cultures have been cultivated under the same light regime, changes in light harvesting capabilities would be the result of changes in levels of light harvesting pigments. However, chlorophyll a measurements revealed similar concentrations in algal cells over a period of 17 days, whether in the presence or absence of bacteria (Table 3). Only after bleaching, when the majority of the algal population was dead, a marked decrease in chlorophyll a concentration was evident in agreement with the macroscopic phenotype of bleaching (Figure 6d) and microscopic phenotype of chloroplast deformation (Figure 6l) that are seen in the co-culture at the same time. Thus, enhanced algal growth does not seem to be the result of more efficient light harvesting. The mechanisms underlying increased algal growth in response to IAA remain unknown.

IAA-induced growth in our system could represent the loss of growth control by E. huxleyi. In various microorganisms, regulatory circuits lead to growth cessation before resources have been completely exhausted. When such controls are bypassed, growth does not stop and higher yields are obtained. Two examples can be discussed in the context of elevated growth as a consequence of loss of growth control; E. coli mutants in rpoS grow to higher cell numbers than the wild type (Vulic and Kolter, 2001). While the wild type senses the near depletion of essential nutrients and prepares in advance by ceasing growth, the mutant lacks this regulatory ability (Vulic and Kolter, 2001). Another example of growth regulation by microorganisms is the ability of cells to sense their critical density, a process referred to as quorum sensing (QS). This process requires the release and sensing of signal molecules that serve as reporters of the population density. The response to increased cell numbers is often a transition to decreased growth, which promotes survival of the cells under conditions of limited resources. In trypanosomes, perturbed ability to sense signal molecules results in failure to undergo growth arrest and leads to uncontrolled proliferatation (Mony and Matthews, 2015). Whether E. huxleyi possesses regulatory circuits to control growth cessation, and whether IAA can influence such regulation, is yet to be explored.

Bacteria in our co-cultures were found attached solely to naked algal cells. We did not detect bacteria attached to calcified algae or shed coccoliths. What underlies the specific bacterial attachment to naked algal cells? Roseobacters posses the ability to chemotax (Miller and Belas, 2006; Miller et al., 2004), thus it is likely that P. inhibens swims in its planktonic stage specifically towards the naked cells. In turn, this might suggest that naked cells release increased levels of exudates. However, the question still remains whether the algal cells are naked irrespective of bacteria, or whether bacteria encourage coccoliths shedding thus increasing the amount of naked algal cells. In every population of calcified E. huxleyi, a sub-population of naked non-calcified cells is seen. It has been previously demonstrated that microzooplankton display higher growth rates when feeding on naked E. huxleyi cells in comparison to calcified cells (Harvey et al., 2015). Thus, bacteria might initially target a pre-existing algal sub-population. Reversible modes of attachment were shown to take place when bacteria such as A. tumefaciens are assessing whether or not to attach to a substrate in a new environment (Heindl et al., 2014). As P. inhibens shares several similarities with this bacterium regarding their mode of attachment (Segev et al., 2015), it is possible that P. inhibens evaluates the algal cells through reversible interactions. In this regard, the physical properties of a naked non-calcified algal cell might be more suitable for bacterial attachment. Similar influences might promote the permanent attachment that persists even after the algal cell has died (Figure 10).

In this study we have established a well-defined microbial model system and carried out a detailed characterization of micro-scale interactions over time. The discovery of a dynamic microbial interaction and the mechanisms underlying it was made possible due to the use of a simplified experimental system for coccolithophore-bacteria interactions. Exploration of similar robust model systems could further reveal chemical and molecular details that might be studied in the ocean. Attempts to link laboratory findings and environmental studies will both widen and deepen our understanding of microbial interactions, their ecophysiology and the extent to which these interactions influence the marine environment.

1. 1. Segev E
   2. Wyche TP
   3. Kim KH
   4. Petersen J
   5. Ellebrandt C
   6. Vlamakis H
   7. Barteneva N
   8. Paulson JN
   9. Chai L
   10. Clardy J
   11. Kolter R

   (2016) Dynamic Metabolic Exchange Governs a Marine Algal-Bacterial Interaction

   Publicly available at the NCBI Short Read Archive (accession no: SRP075256).

   https://www.ncbi.nlm.nih.gov/sra/?term=SRP075256

1. 1. Acinas SG
   2. Antón J
   3. Rodríguez-Valera F

   (1999)

   Diversity of free-living and attached bacteria in offshore western mediterranean waters as depicted by analysis of genes encoding 16S rRNA

   Applied and Environmental Microbiology 65:514–522.

   • PubMed
   • Google Scholar
2. 1. Alcolombri U
   2. Ben-Dor S
   3. Feldmesser E
   4. Levin Y
   5. Tawfik DS
   6. Vardi A

   (2015)  Identification of the algal dimethyl sulfide-releasing enzyme: A missing link in the marine sulfur cycle

   Science 348:1466–1469.

   https://doi.org/10.1126/science.aab1586

   • PubMed
   • Google Scholar
3. 1. Amin SA
   2. Hmelo LR
   3. van Tol HM
   4. Durham BP
   5. Carlson LT
   6. Heal KR
   7. Morales RL
   8. Berthiaume CT
   9. Parker MS
   10. Djunaedi B
   11. Ingalls AE
   12. Parsek MR
   13. Moran MA
   14. Armbrust EV

   (2015) Interaction and signalling between a cosmopolitan phytoplankton and associated bacteria

   Nature 522:98–101.

   https://doi.org/10.1038/nature14488

   • PubMed
   • Google Scholar
4. 1. Balch WM
   2. Drapeau DT
   3. Bowler BC
   4. Huntington TG

   (2012) Step-changes in the physical, chemical and biological characteristics of the Gulf of Maine, as documented by the GNATS time series

   Marine Ecology Progress Series 450:11–35.

   https://doi.org/10.3354/meps09555

   • Google Scholar
5. 1. Balch WM
   2. Drapeau DT
   3. Cucci TL
   4. Vaillancourt RD
   5. Kilpatrick KA
   6. Fritz JJ

   (1999) Optical backscattering by calcifying algae: separating the contribution of particulate inorganic and organic carbon fractions

   Journal of Geophysical Research: Oceans 104:1541–1558.

   https://doi.org/10.1029/1998JC900035

   • Google Scholar
6. 1. Balch WM
   2. Holligan PM
   3. Ackleson SG
   4. Voss KJ

   (1991) Biological and optical properties of mesoscale coccolithophore blooms in the Gulf of Maine

   Limnology and Oceanography 36:629–643.

   https://doi.org/10.4319/lo.1991.36.4.0629

   • Google Scholar
7. 1. Beaufort L
   2. Probert I
   3. de Garidel-Thoron T
   4. Bendif EM
   5. Ruiz-Pino D
   6. Metzl N
   7. Goyet C
   8. Buchet N
   9. Coupel P
   10. Grelaud M
   11. Rost B
   12. Rickaby RE
   13. de Vargas C

   (2011) Sensitivity of coccolithophores to carbonate chemistry and ocean acidification

   Nature 476:80–83.

   https://doi.org/10.1038/nature10295

   • PubMed
   • Google Scholar
8. 1. Behrenfeld MJ
   2. Boss ES

   (2014) Resurrecting the ecological underpinnings of ocean plankton blooms

   Annual Review of Marine Science 6:167–194.

   https://doi.org/10.1146/annurev-marine-052913-021325

   • PubMed
   • Google Scholar
9. 1. Berman-Frank I
   2. Bidle KD
   3. Haramaty L
   4. Falkowski PG

   (2004) The demise of the marine cyanobacterium, Trichodesmium spp., via an autocatalyzed cell death pathway

   Limnology and Oceanography 49:997–1005.

   https://doi.org/10.4319/lo.2004.49.4.0997

   • Google Scholar
10. 1. Bidle KD

    (2015) The molecular ecophysiology of programmed cell death in marine phytoplankton

    Annual Review of Marine Science 7:341–375.

    https://doi.org/10.1146/annurev-marine-010213-135014

    • PubMed
    • Google Scholar
11. 1. Bidle KD

    (2016) Programmed Cell Death in unicellular Phytoplankton

    Current Biology 26:R594–607.

    https://doi.org/10.1016/j.cub.2016.05.056

    • PubMed
    • Google Scholar
12. 1. Bidle KD
    2. Haramaty L
    3. Barcelos E Ramos J
    4. Falkowski P

    (2007) Viral activation and recruitment of metacaspases in the unicellular coccolithophore, Emiliania huxleyi

    PNAS 104:6049–6054.

    https://doi.org/10.1073/pnas.0701240104

    • PubMed
    • Google Scholar
13. 1. Bidle KD
    2. Vardi A

    (2011) A chemical arms race at sea mediates algal host-virus interactions

    Current Opinion in Microbiology 14:449–457.

    https://doi.org/10.1016/j.mib.2011.07.013

    • PubMed
    • Google Scholar
14. 1. Brandl MT
    2. Lindow SE

    (1996)

    Cloning and characterization of a locus encoding an indolepyruvate decarboxylase involved in indole-3-acetic acid synthesis in Erwinia herbicola

    Applied and Environmental Microbiology 62:4121–4128.

    • PubMed
    • Google Scholar
15. 1. Bratbak G
    2. Egge JK
    3. Heldal M

    (1993) Viral mortality of the marine alga Emiliania huxleyi (Haptophyceae) and termination of algal blooms

    Marine Ecology Progress Series 93:39–48.

    https://doi.org/10.3354/meps093039

    • Google Scholar
16. 1. Brock NL
    2. Citron CA
    3. Zell C
    4. Berger M
    5. Wagner-Döbler I
    6. Petersen J
    7. Brinkhoff T
    8. Simon M
    9. Dickschat JS

    (2013) Isotopically labeled sulfur compounds and synthetic selenium and tellurium analogues to study sulfur metabolism in marine bacteria

    Beilstein Journal of Organic Chemistry 9:942–950.

    https://doi.org/10.3762/bjoc.9.108

    • PubMed
    • Google Scholar
17. 1. Buddruhs N
    2. Pradella S
    3. Göker M
    4. Päuker O
    5. Pukall R
    6. Spröer C
    7. Schumann P
    8. Petersen J
    9. Brinkhoff T

    (2013) Molecular and phenotypic analyses reveal the non-identity of the Phaeobacter gallaeciensis type strain deposits CIP 105210T and DSM 17395

    International Journal of Systematic and Evolutionary Microbiology 63:4340–4349.

    https://doi.org/10.1099/ijs.0.053900-0

    • PubMed
    • Google Scholar
18. 1. Charlson RJ
    2. Lovelock JE
    3. Andreae MO
    4. Warren SG

    (1987) Oceanic phytoplankton, atmospheric sulphur, cloud albedo and climate

    Nature 326:655–661.

    https://doi.org/10.1038/326655a0

    • Google Scholar
19. 1. Costacurta A
    2. Vanderleyden J

    (1995) Synthesis of phytohormones by plant-associated bacteria

    Critical Reviews in Microbiology 21:1–18.

    https://doi.org/10.3109/10408419509113531

    • PubMed
    • Google Scholar
20. 1. Curson AR
    2. Todd JD
    3. Sullivan MJ
    4. Johnston AW

    (2011) Catabolism of dimethylsulphoniopropionate: microorganisms, enzymes and genes

    Nature Reviews Microbiology 9:849–859.

    https://doi.org/10.1038/nrmicro2653

    • PubMed
    • Google Scholar
21. 1. Durham BP
    2. Sharma S
    3. Luo H
    4. Smith CB
    5. Amin SA
    6. Bender SJ
    7. Dearth SP
    8. Van Mooy BA
    9. Campagna SR
    10. Kujawinski EB
    11. Armbrust EV
    12. Moran MA

    (2015) Cryptic carbon and sulfur cycling between surface ocean plankton

    PNAS 112:453–457.

    https://doi.org/10.1073/pnas.1413137112

    • PubMed
    • Google Scholar
22. 1. Falkowski PG
    2. Dubinsky Z
    3. Wyman K

    (1985) Growth-irradiance relationships in phytoplankton

    Limnology and Oceanography 30:311–321.

    https://doi.org/10.4319/lo.1985.30.2.0311

    • Google Scholar
23. 1. Field CB
    2. Behrenfeld MJ
    3. Randerson JT
    4. Falkowski P

    (1998) Primary production of the biosphere: integrating terrestrial and oceanic components

    Science 281:237–240.

    https://doi.org/10.1126/science.281.5374.237

    • PubMed
    • Google Scholar
24. 1. Fiore CL
    2. Longnecker K
    3. Kido Soule MC
    4. Kujawinski EB

    (2015) Release of ecologically relevant metabolites by the cyanobacterium Synechococcus elongates CCMP 1631

    Environmental Microbiology 17:3949–3963.

    https://doi.org/10.1111/1462-2920.12899

    • PubMed
    • Google Scholar
25. 1. Frank O
    2. Michael V
    3. Päuker O
    4. Boedeker C
    5. Jogler C
    6. Rohde M
    7. Petersen J

    (2015) Plasmid curing and the loss of grip--the 65-kb replicon of Phaeobacter inhibens DSM 17395 is required for biofilm formation, motility and the colonization of marine algae

    Systematic and Applied Microbiology 38:120–127.

    https://doi.org/10.1016/j.syapm.2014.12.001

    • PubMed
    • Google Scholar
26. 1. Fulton JM
    2. Fredricks HF
    3. Bidle KD
    4. Vardi A
    5. Kendrick BJ
    6. DiTullio GR
    7. Van Mooy BA

    (2014) Novel molecular determinants of viral susceptibility and resistance in the lipidome of Emiliania huxleyi

    Environmental Microbiology 16:1137–1149.

    https://doi.org/10.1111/1462-2920.12358

    • PubMed
    • Google Scholar
27. 1. Gavrieli Y
    2. Sherman Y
    3. Ben-Sasson SA

    (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation

    The Journal of Cell Biology 119:493–501.

    https://doi.org/10.1083/jcb.119.3.493

    • PubMed
    • Google Scholar
28. 1. González JM
    2. Kiene RP
    3. Moran MA

    (1999)

    Transformation of sulfur compounds by an abundant lineage of marine bacteria in the alpha-subclass of the class Proteobacteria

    Applied and Environmental Microbiology 65:3810–3819.

    • PubMed
    • Google Scholar
29. 1. González JM
    2. Simó R
    3. Massana R
    4. Covert JS
    5. Casamayor EO
    6. Pedrós-Alió C
    7. Moran MA
    8. Simo R
    9. Pedros-Alio C

    (2000) Bacterial community structure associated with a dimethylsulfoniopropionate-producing North Atlantic algal bloom

    Applied and Environmental Microbiology 66:4237–4246.

    https://doi.org/10.1128/AEM.66.10.4237-4246.2000

    • PubMed
    • Google Scholar
30. 1. Green DH
    2. Echavarri-Bravo V
    3. Brennan D
    4. Hart MC

    (2015) Bacterial diversity associated with the coccolithophorid algae Emiliania huxleyi and Coccolithus pelagicus f. braarudii

    BioMed Research International 2015:194540.

    https://doi.org/10.1155/2015/194540

    • PubMed
    • Google Scholar
31. 1. Guillard RRL
    2. Hargraves PE

    (1993) Stichochrysis immobilis is a diatom, not a chrysophyte

    Phycologia 32:234–236.

    https://doi.org/10.2216/i0031-8884-32-3-234.1

    • Google Scholar
32. 1. Harvey EL
    2. Bidle KD
    3. Johnson MD

    (2015) Consequences of strain variability and calcification in Emiliania huxleyi on microzooplankton grazing

    Journal of Plankton Research 37:fbv081.

    https://doi.org/10.1093/plankt/fbv081

    • Google Scholar
33. 1. Harvey EL
    2. Deering RW
    3. Rowley DC
    4. El Gamal A
    5. Schorn M
    6. Moore BS
    7. Johnson MD
    8. Mincer TJ
    9. Whalen KE

    (2016) A Bacterial Quorum-sensing precursor induces mortality in the marine coccolithophore, Emiliania huxleyi

    Frontiers in Microbiology 7:59.

    https://doi.org/10.3389/fmicb.2016.00059

    • PubMed
    • Google Scholar
34. 1. Heindl JE
    2. Wang Y
    3. Heckel BC
    4. Mohari B
    5. Feirer N
    6. Fuqua C

    (2014) Mechanisms and regulation of surface interactions and biofilm formation in Agrobacterium

    Frontiers in Plant Science 5:176.

    https://doi.org/10.3389/fpls.2014.00176

    • PubMed
    • Google Scholar
35. 1. Holligan PM
    2. Viollier M
    3. Harbour DS
    4. Camus P
    5. Champagne-Philippe M

    (1983) Satellite and ship studies of coccolithophore production along a continental shelf edge

    Nature 304:339–342.

    https://doi.org/10.1038/304339a0

    • Google Scholar
36. 1. Iglesias-Rodríguez MD
    2. Brown CW
    3. Doney SC
    4. Kleypas J
    5. Kolber D
    6. Kolber Z
    7. Hayes PK
    8. Falkowski PG

    (2002) Representing key phytoplankton functional groups in ocean carbon cycle models: Coccolithophorids

    Global Biogeochemical Cycles 16:47-1–47-20.

    https://doi.org/10.1029/2001GB001454

    • Google Scholar
37. 1. Johnson JG
    2. Janech MG
    3. Van Dolah FM

    (2014) Caspase-like activity during aging and cell death in the toxic dinoflagellate Karenia brevis

    Harmful Algae 31:41–53.

    https://doi.org/10.1016/j.hal.2013.08.005

    • Google Scholar
38. 1. Johnson WM
    2. Kido Soule MC
    3. Kujawinski EB

    (2016) Evidence for quorum sensing and differential metabolite production by a marine bacterium in response to DMSP

    The ISME Journal 10:2304–2316.

    https://doi.org/10.1038/ismej.2016.6

    • PubMed
    • Google Scholar
39. 1. Kepinski S
    2. Leyser O

    (2005) The Arabidopsis F-box protein TIR1 is an auxin receptor

    Nature 435:446–451.

    https://doi.org/10.1038/nature03542

    • PubMed
    • Google Scholar
40. 1. Labeeuw L
    2. Khey J
    3. Bramucci AR
    4. Atwal H
    5. de la Mata AP
    6. Harynuk J
    7. Case RJ

    (2016) Indole-3-acetic Acid is produced by Emiliania huxleyi coccolith-bearing cells and triggers a physiological response in bald Cells

    Frontiers in Microbiology 7:828.

    https://doi.org/10.3389/fmicb.2016.00828

    • PubMed
    • Google Scholar
41. 1. Lehahn Y
    2. Koren I
    3. Schatz D
    4. Frada M
    5. Sheyn U
    6. Boss E
    7. Efrati S
    8. Rudich Y
    9. Trainic M
    10. Sharoni S
    11. Laber C
    12. DiTullio GR
    13. Coolen MJ
    14. Martins AM
    15. Van Mooy BA
    16. Bidle KD
    17. Vardi A

    (2014) Decoupling physical from biological processes to assess the impact of viruses on a mesoscale algal bloom

    Current Biology 24:2041–2046.

    https://doi.org/10.1016/j.cub.2014.07.046

    • PubMed
    • Google Scholar
42. 1. Marsh ME

    (2003) Regulation of CaCO₃ formation in coccolithophores

    Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 136:743–754.

    https://doi.org/10.1016/S1096-4959(03)00180-5

    • Google Scholar
43. 1. Mayali X
    2. Azam F

    (2004) Algicidal bacteria in the sea and their impact on algal blooms

    The Journal of Eukaryotic Microbiology 51:139–144.

    https://doi.org/10.1111/j.1550-7408.2004.tb00538.x

    • PubMed
    • Google Scholar
44. 1. Miller TR
    2. Belas R

    (2004) Dimethylsulfoniopropionate metabolism by Pfiesteria-associated Roseobacter spp

    Applied and Environmental Microbiology 70:3383–3391.

    https://doi.org/10.1128/AEM.70.6.3383-3391.2004

    • PubMed
    • Google Scholar
45. 1. Miller TR
    2. Belas R

    (2006) Motility is involved in Silicibacter sp. TM1040 interaction with dinoflagellates

    Environmental Microbiology 8:1648–1659.

    https://doi.org/10.1111/j.1462-2920.2006.01071.x

    • PubMed
    • Google Scholar
46. 1. Miller TR
    2. Hnilicka K
    3. Dziedzic A
    4. Desplats P
    5. Belas R

    (2004) Chemotaxis of Silicibacter sp. strain TM1040 toward dinoflagellate products

    Applied and Environmental Microbiology 70:4692–4701.

    https://doi.org/10.1128/AEM.70.8.4692-4701.2004

    • PubMed
    • Google Scholar
47. 1. Mony BM
    2. Matthews KR

    (2015) Assembling the components of the quorum sensing pathway in african trypanosomes

    Molecular Microbiology 96:220–232.

    https://doi.org/10.1111/mmi.12949

    • PubMed
    • Google Scholar
48. 1. Moran MA
    2. Belas R
    3. Schell MA
    4. González JM
    5. Sun F
    6. Sun S
    7. Binder BJ
    8. Edmonds J
    9. Ye W
    10. Orcutt B
    11. Howard EC
    12. Meile C
    13. Palefsky W
    14. Goesmann A
    15. Ren Q
    16. Paulsen I
    17. Ulrich LE
    18. Thompson LS
    19. Saunders E
    20. Buchan A

    (2007) Ecological genomics of marine roseobacters

    Applied and Environmental Microbiology 73:4559–4569.

    https://doi.org/10.1128/AEM.02580-06

    • PubMed
    • Google Scholar
49. 1. O'Toole GA
    2. Kolter R

    (1998) Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis

    Molecular Microbiology 28:449–461.

    https://doi.org/10.1046/j.1365-2958.1998.00797.x

    • PubMed
    • Google Scholar
50. 1. Ondov BD
    2. Bergman NH
    3. Phillippy AM

    (2011) Interactive metagenomic visualization in a web browser

    BMC Bioinformatics 12:385.

    https://doi.org/10.1186/1471-2105-12-385

    • PubMed
    • Google Scholar
51. 1. Paasche E

    (2001) A review of the coccolithophorid Emiliania huxleyi (Prymnesiophyceae), with particular reference to growth, coccolith formation, and calcification-photosynthesis interactions

    Phycologia 40:503–529.

    https://doi.org/10.2216/i0031-8884-40-6-503.1

    • Google Scholar
52. 1. Patten CL
    2. Glick BR

    (2002) Role of Pseudomonas putida indoleacetic acid in development of the host plant root system

    Applied and Environmental Microbiology 68:3795–3801.

    https://doi.org/10.1128/AEM.68.8.3795-3801.2002

    • PubMed
    • Google Scholar
53. 1. Persello-Cartieaux F
    2. David P
    3. Sarrobert C
    4. Thibaud MC
    5. Achouak W
    6. Robaglia C
    7. Nussaume L

    (2001) Utilization of mutants to analyze the interaction between Arabidopsis thaliana and its naturally root-associated Pseudomonas

    Planta 212:190–198.

    https://doi.org/10.1007/s004250000384

    • PubMed
    • Google Scholar
54. 1. Persello-cartieaux F
    2. Nussaume L
    3. Robaglia C

    (2003) Tales from the underground: molecular . plant-Rhizobacteria interactions

     Plant, Cell and Environment 26:189–199.

    https://doi.org/10.1046/j.1365-3040.2003.00956.x

    • Google Scholar
55. 1. Petersen J
    2. Brinkmann H
    3. Berger M
    4. Brinkhoff T
    5. Päuker O
    6. Pradella S

    (2011) Origin and evolution of a novel DnaA-like plasmid replication type in Rhodobacterales

    Molecular Biology and Evolution 28:1229–1240.

    https://doi.org/10.1093/molbev/msq310

    • PubMed
    • Google Scholar
56. 1. Ponomarev ED
    2. Veremeyko T
    3. Barteneva NS

    (2011) Visualization and quantitation of the expression of microRNAs and their target genes in neuroblastoma single cells using imaging cytometry

    BMC Research Notes 4:517.

    https://doi.org/10.1186/1756-0500-4-517

    • PubMed
    • Google Scholar
57. 1. Prinsen E

    (1993) Azospirillum brasilense Indole-3-Acetic acid biosynthesis: evidence for a non-tryptophan dependent pathway

    Molecular Plant-Microbe Interactions 6:609–615.

    https://doi.org/10.1094/MPMI-6-609

    • Google Scholar
58. 1. Quinn PK
    2. Bates TS

    (2011) The case against climate regulation via oceanic phytoplankton sulphur emissions

    Nature 480:51–56.

    https://doi.org/10.1038/nature10580

    • PubMed
    • Google Scholar
59. 1. Rohwer F
    2. Thurber RV

    (2009) Viruses manipulate the marine environment

    Nature 459:207–212.

    https://doi.org/10.1038/nature08060

    • PubMed
    • Google Scholar
60. 1. Sabine CL
    2. Feely RA
    3. Gruber N
    4. Key RM
    5. Lee K
    6. Bullister JL
    7. Wanninkhof R
    8. Wong CS
    9. Wallace DW
    10. Tilbrook B
    11. Millero FJ
    12. Peng TH
    13. Kozyr A
    14. Ono T
    15. Rios AF

    (2004) The oceanic sink for anthropogenic CO₂

    Science 305:367–371.

    https://doi.org/10.1126/science.1097403

    • PubMed
    • Google Scholar
61. 1. Segev E
    2. Castañeda IS
    3. Sikes EL
    4. Vlamakis H
    5. Kolter R

    (2016) Bacterial influence on alkenones in live microalgae

    Journal of Phycology 52:125–130.

    https://doi.org/10.1111/jpy.12370

    • PubMed
    • Google Scholar
62. 1. Segev E
    2. Tellez A
    3. Vlamakis H
    4. Kolter R

    (2015) Morphological heterogeneity and attachment of Phaeobacter inhibens

    PLoS One 10:e0141300.

    https://doi.org/10.1371/journal.pone.0141300

    • PubMed
    • Google Scholar
63. 1. Segovia M
    2. Haramaty L
    3. Berges JA
    4. Falkowski PG

    (2003) Cell death in the unicellular chlorophyte Dunaliella tertiolecta. A hypothesis on the evolution of apoptosis in higher plants and metazoans

    Plant Physilogy 132:99–105.

    https://doi.org/10.1104/pp.102.017129

    • Google Scholar
64. 1. Seyedsayamdost MR
    2. Case RJ
    3. Kolter R
    4. Clardy J

    (2011) The Jekyll-and-Hyde chemistry of Phaeobacter gallaeciensis

    Nature Chemistry 3:331–335.

    https://doi.org/10.1038/nchem.1002

    • PubMed
    • Google Scholar
65. 1. Seymour JR
    2. Simó R
    3. Ahmed T
    4. Stocker R

    (2010) Chemoattraction to dimethylsulfoniopropionate throughout the marine microbial food web

    Science 329:342–345.

    https://doi.org/10.1126/science.1188418

    • PubMed
    • Google Scholar
66. 1. Sheyn U
    2. Rosenwasser S
    3. Ben-Dor S
    4. Porat Z
    5. Vardi A

    (2016) Modulation of host ROS metabolism is essential for viral infection of a bloom-forming coccolithophore in the ocean

    The ISME Journal 10:1742–1754.

    https://doi.org/10.1038/ismej.2015.228

    • PubMed
    • Google Scholar
67. 1. Simó R

    (2001) Production of atmospheric sulfur by oceanic plankton: biogeochemical, ecological and evolutionary links

    Trends in Ecology & Evolution 16:287–294.

    https://doi.org/10.1016/S0169-5347(01)02152-8

    • PubMed
    • Google Scholar
68. 1. Smriga S
    2. Fernandez VI
    3. Mitchell JG
    4. Stocker R

    (2016) Chemotaxis toward phytoplankton drives organic matter partitioning among marine bacteria

    PNAS 113:1576–1581.

    https://doi.org/10.1073/pnas.1512307113

    • PubMed
    • Google Scholar
69. 1. Spaepen S
    2. Vanderleyden J
    3. Remans R

    (2007) Indole-3-acetic acid in microbial and microorganism-plant signaling

    FEMS Microbiology Reviews 31:425–448.

    https://doi.org/10.1111/j.1574-6976.2007.00072.x

    • PubMed
    • Google Scholar
70. 1. Subramoni S
    2. Nathoo N
    3. Klimov E
    4. Yuan ZC

    (2014) Agrobacterium tumefaciens responses to plant-derived signaling molecules

    Frontiers in Plant Science 5:322.

    https://doi.org/10.3389/fpls.2014.00322

    • PubMed
    • Google Scholar
71. 1. Sunda W
    2. Kieber DJ
    3. Kiene RP
    4. Huntsman S

    (2002) An antioxidant function for DMSP and DMS in marine algae

    Nature 418:317–320.

    https://doi.org/10.1038/nature00851

    • PubMed
    • Google Scholar
72. 1. Theunis M
    2. Kobayashi H
    3. Broughton WJ
    4. Prinsen E

    (2004) Flavonoids, NodD1, NodD2, and nod-box NB15 modulate expression of the y4wEFG locus that is required for indole-3-acetic acid synthesis in Rhizobium sp. strain NGR234

    Molecular Plant-Microbe Interactions 17:1153–1161.

    https://doi.org/10.1094/MPMI.2004.17.10.1153

    • PubMed
    • Google Scholar
73. 1. Tyrrell T
    2. Merico A

    (2004)

    Coccolithophores From Molecular Processes to Global Impact

    75–97, Emiliania huxleyi: bloom observations and the conditions that induce them, Coccolithophores From Molecular Processes to Global Impact, Berlin, Springer.

    • Google Scholar
74. 1. Vardi A
    2. Haramaty L
    3. Van Mooy BA
    4. Fredricks HF
    5. Kimmance SA
    6. Larsen A
    7. Bidle KD

    (2012) Host-virus dynamics and subcellular controls of cell fate in a natural coccolithophore population

    PNAS 109:19327–19332.

    https://doi.org/10.1073/pnas.1208895109

    • PubMed
    • Google Scholar
75. 1. Vardi A
    2. Van Mooy BA
    3. Fredricks HF
    4. Popendorf KJ
    5. Ossolinski JE
    6. Haramaty L
    7. Bidle KD

    (2009) Viral glycosphingolipids induce lytic infection and cell death in marine phytoplankton

    Science 326:861–865.

    https://doi.org/10.1126/science.1177322

    • PubMed
    • Google Scholar
76. 1. Vulic M
    2. Kolter R

    (2001)

    Evolutionary cheating in Escherichia coli stationary phase cultures

    Genetics 158:519–526.

    • PubMed
    • Google Scholar
77. 1. Wang H
    2. Tomasch J
    3. Jarek M
    4. Wagner-Döbler I

    (2014) A dual-species co-cultivation system to study the interactions between Roseobacters and dinoflagellates

    Frontiers in Microbiology 5:311.

    https://doi.org/10.3389/fmicb.2014.00311

    • PubMed
    • Google Scholar
78. 1. Wang R
    2. Gallant É
    3. Seyedsayamdost MR

    (2016) Investigation of the genetics and biochemistry of roseobacticide production in the roseobacter clade bacterium Phaeobacter inhibens

    mBio 7:e02118.

    https://doi.org/10.1128/mBio.02118-15

    • PubMed
    • Google Scholar
79. 1. Wood DE
    2. Salzberg SL

    (2014) Kraken: ultrafast metagenomic sequence classification using exact alignments

    Genome Biology 15:R46.

    https://doi.org/10.1186/gb-2014-15-3-r46

    • PubMed
    • Google Scholar
80. 1. Xie H
    2. Pasternak JJ
    3. Glick BR

    (1996) Isolation and characterization of mutants of the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2 that overproduce indoleacetic acid

    Current Microbiology 32:67–71.

    https://doi.org/10.1007/s002849900012

    • Google Scholar
81. 1. Xu J
    2. Kim J
    3. Koestler BJ
    4. Choi JH
    5. Waters CM
    6. Fuqua C

    (2013) Genetic analysis of Agrobacterium tumefaciens unipolar polysaccharide production reveals complex integrated control of the motile-to-sessile switch

    Molecular Microbiology 89:929–948.

    https://doi.org/10.1111/mmi.12321

    • PubMed
    • Google Scholar
82. 1. Zech H
    2. Thole S
    3. Schreiber K
    4. Kalhöfer D
    5. Voget S
    6. Brinkhoff T
    7. Simon M
    8. Schomburg D
    9. Rabus R

    (2009) Growth phase-dependent global protein and metabolite profiles of Phaeobacter gallaeciensis strain DSM 17395, a member of the marine Roseobacter-clade

    Proteomics 9:3677–3697.

    https://doi.org/10.1002/pmic.200900120

    • PubMed
    • Google Scholar
83. 1. Zimmer W
    2. Wesche M
    3. Timmermans L

    (1998) Identification and isolation of the indole-3-pyruvate decarboxylase gene from Azospirillum brasilense Sp7: sequencing and functional analysis of the gene locus

    Current Microbiology 36:327–331.

    https://doi.org/10.1007/s002849900317

    • PubMed
    • Google Scholar

Author details

1. Einat Segev

   Department of Microbiology and Immunobiology, Harvard Medical School, Boston, United States

   Contribution

   ES, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   Einat_Segev@hms.harvard.edu

   Competing interests

   No competing interests declared.

   "This ORCID iD identifies the author of this article:" 0000-0002-2266-1219

2. Thomas P Wyche

   Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States

   Contribution

   TPW, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

3. Ki Hyun Kim

   Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States

   Present address

   School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea

   Contribution

   KHK, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

   "This ORCID iD identifies the author of this article:" 0000-0002-5285-9138

4. Jörn Petersen

   Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany

   Contribution

   JP, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   No competing interests declared.

5. Claire Ellebrandt

   Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany

   Contribution

   CE, Acquisition of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   No competing interests declared.

6. Hera Vlamakis

   Department of Microbiology and Immunobiology, Harvard Medical School, Boston, United States

   Present address

   Broad Institute, Cambridge, United States

   Contribution

   HV, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

7. Natasha Barteneva

   Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, United States

   Contribution

   NB, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

8. Joseph N Paulson

   Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, United States

   Contribution

   JNP, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

9. Liraz Chai

   Department of Microbiology and Immunobiology, Harvard Medical School, Boston, United States

   Present address

   Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel

   Contribution

   LC, Acquisition of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   No competing interests declared.

10. Jon Clardy

    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States

    Contribution

    JC, Conception and design, Analysis and interpretation of data, Drafting or revising the article

    Competing interests

    JC: Reviewing editor, eLife

    "This ORCID iD identifies the author of this article:" 0000-0003-0213-8356

11. Roberto Kolter

    Department of Microbiology and Immunobiology, Harvard Medical School, Boston, United States

    Contribution

    RK, Conception and design, Analysis and interpretation of data, Drafting or revising the article

    For correspondence

    roberto_kolter@hms.harvard.edu

    Competing interests

    No competing interests declared.

    "This ORCID iD identifies the author of this article:" 0000-0001-9548-1481

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