Little is known about AAP bacteria in polar environments. The main objective of this study was to explore their abundance, diversity, and distribution patterns in a significant and typical arctic region and to evaluate their ecology and biogeographical trends. Our results indicate that the distribution patterns generally observed in oligotrophic open ocean and eutrophic estuarine environments of temperate and tropical regions (Jiao et al., 2007; Waidner & Kirchman, 2008; Lamy et al., 2011) also occur in the Beaufort Sea.
Links between AAP population abundance and environmental variables
Our results indicate that particulate and dissolved organic matter and Chl a concentrations positively influenced the relative abundance of AAP bacteria, but did not contribute to the success of the prokaryote community as a whole. This suggests that AAP bacteria may respond to organic supply differently and have higher mineralization capacities than the bacterial community. In addition to AAP bacteria associated with the thick surface turbidity layer, proportions of AAP bacteria were high in the thinner but more turbid BNL. Interestingly, AAP bacteria in surface and bottom layers showed contrasting relationships to several environmental variables, suggesting that the two particle-rich environments represent substantially different habitats. The two habitats could differ because of different types of particles in the water column, phytoplankton, and mineral-rich fine particles in the surface layer and coarse aggregates of suspended sediments in the bottom layer (Doxaran et al., 2012). These particle-rich waters might be important not only in terms of sediment transport but also as sites of microbially mediated organic transformations. The latter possibility is consistent with observations of increased microbial production and activity in particle-rich surface and bottom waters of the Mackenzie plume (Vallières et al., 2008; Garneau et al., 2009). Although we did not quantify the proportion of particle-associated AAP bacteria, earlier studies in other nutrient-rich environments (Waidner & Kirchman, 2007) suggest that they may represent a significant part of the AAP bacterial community in the Mackenzie plume. Being associated with particles seems to enhance the synthesis of BChl a in estuaries (Cottrell et al., 2010). It is likely that the energy gained by AAP bacteria via phototrophy in these environments is suitable for cost-intensive metabolic processes such as degradation of humic-rich DOM from riverine and terrestrial runoff.
Aerobic anoxygenic phototrophic bacterial diversity and distribution
Most OTUs recovered were closely related (> 94% similarity) to existing Arctic sequences or belonged to novel OTUs. Only 8 OTUs matched sequences from elsewhere. The arctic pufM genes were also different from those in Antarctic sea ice and coastal seawater (Koh et al., 2011). Consistent with previous findings (Cottrell & Kirchman, 2009), this suggests that most OTUs found in this study might be restricted to the Beaufort Sea, which seems to constitute a microbial province favoring endemism (Lovejoy et al., 2007).
The most striking observation from our dataset is the widespread distribution of a AAP betaproteobacterial clade (OTU 6) in the entire shelf. Betaproteobacteria are usually low in abundance in the open ocean and the few betaproteobacterial sequences that have been retrieved from the marine environment are from coastal environments (Rappé et al., 2000; Riemann et al., 2008). In contrast, they represent a consistently large fraction of the bacterioplankton in freshwater lakes and diverse river types (Glöckner et al., 2000; Zwart et al., 2002) including the Mackenzie River where they are abundant (Garneau et al., 2006; Galand et al., 2008). Since the Arctic Ocean receives about 10% of global riverine discharge (Aagaard & Carmack, 1989), the stratified surface waters of the Arctic Ocean share many characteristics of an estuary where Betaproteobacteria form a minor fraction of the total prokaryotic community (Galand et al., 2009; Kirchman et al., 2010). Most betaproteobacterial OTUs recovered in this study were new (< 94% similarity to existing sequences) and do not group with the Rhodoferax, Roseateles, and Rubrivivax pufM clusters often identified in other estuarine and freshwater systems (Waidner & Kirchman, 2008; Salka et al., 2011). They formed several clusters divergent from the phylogroup I (Yutin et al., 2007) that include arctic sequences (Cottrell & Kirchman, 2009) and single amplified genome (SAG) sequences from temperate freshwater lakes (Martinez-Garcia et al., 2012). Interestingly, the 16S rRNA sequences from the latter pufM-containing SAGs were primarily related to the betaproteobacterial Polynucleobacter cluster that are common in the Mackenzie river (Galand et al., 2008). As horizontal gene transfer of the photosynthetic gene cluster is possible (Igarashi et al., 2001), phylogenetic data need to be interpreted with caution especially in groups with only a few species with known pufM and 16S sequences.
Our data clearly indicate that betaproteobacterial AAP bacteria exhibited a strong river to ocean gradient, suggesting that these bacteria grew in the Mackenzie River and then were mixed with Beaufort coastal waters. The distribution of OTU 6 in the Beaufort Sea was consistent with satellite observations of the offshore extension of Mackenzie turbid waters over the continental shelf (Doxaran et al., 2012). However, the presence and activity of OTU 6 in Beaufort coastal waters contrasted with that of other betaproteobacterial OTUs found only near the mouth of the Mackenzie River. As mortality of freshwater bacteria can be an important process in estuaries (Painchaud et al., 1995), one explanation is that OTU 6 members have a broader salinity tolerance than other AAP Betaproteobacteria. The presence of partial pufM sequences identical to that of OTU 6 in marine waters of Monterey Bay (Béjà et al., 2002) supports this hypothesis. Further studies are required to estimate their contribution in other marine Arctic regions and to identify parameters that control their distribution.
Previous studies showed that some Betaproteobacteria are still active in marine waters (Cottrell & Kirchman, 2004), including in the Arctic (Alonso-Sáez et al., 2008). Here, we showed that the OTU 6 pufM gene was actively transcribed in saline waters during the arctic summer, which is surprising because BChl a synthesis is partly or totally inhibited by light in AAP bacteria currently in culture (Yurkov & van Gemerden, 1993; Tomasch et al., 2011). Our data suggest that BChl a synthesis in OTU 6 members is not fully repressed by light, like in Dinoroseobacter shibae and Roseateles depolymerans (Suyama et al., 2002; Tomasch et al., 2011). In most AAP bacteria isolated so far, photoheterotrophy generates additional metabolic energy enhancing AAP bacterial growth under light (Koblížek et al., 2003; Cooney et al., 2006). Considering the variety of sharp environmental gradients encountered by OTU 6 members in the transition zone between freshwater and marine waters, it is likely that, when light is sufficient, photoheterotrophy may have different physiological roles in warm, organic matter-rich freshwater and in cold, saline oligotrophic waters. The nature of their physiological adaptation and metabolic versatility needs to be elucidated to understand the selective advantage that photoheterotrophy provides them.
To our knowledge, AAP strains have not previously been isolated from the Arctic Ocean. In this study, the isolates provided a valuable set of data that allowed the taxonomic identification of two predominant pufM phylotypes. We successfully isolated pufM-containing bacteria, but only from surface waters, consistent with the phototrophic character of AAP bacteria. Our arctic isolates were restricted to different species of the genera Sulfitobacter and Loktanella. Although these species were cultivated from temperate to polar regions (Labrenz et al., 2000; Van Trappen et al., 2004; Salka et al., 2008), the strains isolated in this study were most similar to microorganisms residing in polar waters. Among them, the Sulfitobacter strains clustering into OTU 5 were absent outside the Beaufort Sea, suggesting that they potentially represent typical arctic members. Sulfitobacter and Loktanella species are often found in surface waters, and numerous interactions with phytoplankton have been reported (Moran et al., 2007). These metabolically versatile bacteria can satisfy a significant part of their carbon and sulfur demands by assimilating DMSP released during the decay of phytoplankton blooms (González et al., 1999; Mou et al., 2005). The Beaufort Sea waters sampled during this study exhibited postbloom characteristics, with low levels of Chl a in the surface layer. As surface waters of the Beaufort Sea are oligotrophic in summer, the capacity to derive energy from light and reduced sulfur compounds would give a physiological advantage for competing in this extreme environment. The influence of light and starvation on BChl a and biomass formation has been studied in AAP bacteria such as Sulfitobacter and Loktanella that contain low amounts of BChl a (Biebl & Wagner-Döbler, 2006). As this type of AAP bacteria seems to use phototrophy genes only under simultaneous illumination and extreme shortage of organic nutrients, their distribution in arctic waters may be linked to their photoheterotrophic mode of metabolism.
Aerobic anoxygenic phototrophic diversity was highest at the shelf and offshore DCM layers of the Beaufort Sea where Pacific Summer Water mixes with the BNL. Methylobacterium- and Sphingomonas-like bacteria, mostly absent in surface waters, were common at these depths along the Mackenzie plume. Although a few species of both genera have been isolated from seawater (Vancanneyt et al., 2001; Wang et al., 2007), these bacteria have a widespread distribution in diverse terrestrial habitats including soil, freshwater, and lake sediments (White et al., 1996; Green, 2006). Our data suggest that Methylobacterium- and Sphingomonas-like bacteria share the same habitat, more likely associated with soluble humic material in river water (Kirk, 2011). In line with this hypothesis, these bacteria were found to actively participate in humic matter degradation (Balkwill et al., 2006; Hutalle-Schmelzer et al., 2010). Two groups of Bradyrhizobiaceae-related sequences were detected mostly in DCM samples of the Beaufort Sea shelf. One group was loosely associated to the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris that is widely distributed in nature including coastal marine sediments (Oda et al., 2003). The other grouped with strictly AAP Bradyrhizobium strains that form nitrogen-fixing nodules on stems and/or roots of many legumes (Giraud & Fleischman, 2004). Like Methylobacterium and Sphingomonas clusters, these clades were mostly recovered in the deep layers of the Mackenzie plume, suggesting that they were also entrained by the river sediment load. However, their distribution was similar to that of Citromicrobium-like clades that are typical marine bacteria (Yurkov et al., 1999; Jeanthon et al., 2011). Furthermore, we also retrieved them from North Pacific Ocean surface waters, supporting earlier reports of Bradyrhizobiaceae in the North Pacific Ocean gyre and the Bering Sea where terrestrial inputs are unlikely (Hu et al., 2006). It is therefore tempting to speculate that arctic Bradyrhizobiaceae-related pufM sequences are marine photosynthetic bacteria possibly transported in Pacific waters.