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Aerobic anoxygenic phototrophic (AAP) bacteria harvest light energy using bacteriochlorophyll-containing reaction centers to supplement their mostly heterotrophic metabolism. While their abundance and growth have been intensively studied in coastal environments, much less is known about their activity in oligotrophic open ocean regions. Therefore, we combined in situ sampling in the North Pacific Subtropical Gyre, north of O'ahu island, Hawaii, with two manipulation experiments. Infra-red epifluorescence microscopy documented that AAP bacteria represented approximately 2% of total bacteria in the euphotic zone with the maximum abundance in the upper 50 m. They conducted active photosynthetic electron transport with maximum rates up to 50 electrons per reaction center per second. The in situ decline of bacteriochlorophyll concentration over the daylight period, an estimate of loss rates due to predation, indicated that the AAP bacteria in the upper 50 m of the water column turned over at rates of 0.75-0.90 d -1 . This corresponded well with the specific growth rate determined in dilution experiments where AAP bacteria grew at a rate 1.05 ± 0.09 d -1 . An amendment of inorganic nitrogen to obtain N:P = 32 resulted in a more than 10 times increase in AAP abundance over 6 days. The presented data document that AAP bacteria are an active part of the bacterioplankton community in the oligotrophic North Pacific Subtropical Gyre and that their growth was mostly controlled by nitrogen availability and grazing pressure.IMPORTANCEMarine bacteria represent a complex assembly of species with different physiology, metabolism, and substrate preferences. We focus on a specific functional group of marine bacteria called aerobic anoxygenic phototrophs. These photoheterotrophic organisms require organic carbon substrates for growth, but they can also supplement their metabolic needs with light energy captured by bacteriochlorophyll. These bacteria have been intensively studied in coastal regions, but rather less is known about their distribution, growth, and mortality in the oligotrophic open ocean. Therefore, we conducted a suite of measurements in the North Pacific Subtropical Gyre to determine the distribution of these organisms in the water column and their growth and mortality rates. A nutrient amendment experiment showed that aerobic anoxygenic phototrophs were limited by inorganic nitrogen. Despite this, they grew more rapidly than average heterotrophic bacteria, but their growth was balanced by intense grazing pressure., Competing Interests: The authors declare no conflict of interest.

High-throughput sequencing (HTS) of the 16S rRNA gene has been used successfully to describe the structure and dynamics of microbial communities. Picocyanobacteria are important members of bacterioplankton communities, and, so far, they have predominantly been targeted using universal bacterial primers, providing a limited resolution of the picocyanobacterial community structure and dynamics. To increase such resolution, the study of a particular target group is best approached with the use of specific primers. Here, we aimed to design and evaluate specific primers for aquatic picocyanobacterial genera to be used with high-throughput sequencing. Since the various regions of the 16S rRNA gene have different degrees of conservation in different bacterial groups, we therefore first determined which hypervariable region of the 16S rRNA gene provides the highest taxonomic and phylogenetic resolution for the genera Synechococcus , Prochlorococcus , and Cyanobium An in silico analysis showed that the V5, V6, and V7 hypervariable regions appear to be the most informative for this group. We then designed primers flanking these hypervariable regions and tested them in natural marine and freshwater communities. We successfully detected that most (97%) of the obtained reads could be assigned to picocyanobacterial genera. We defined operational taxonomic units as exact sequence variants (zero-radius operational taxonomic units [zOTUs]), which allowed us to detect higher genetic diversity and infer ecologically relevant information about picocyanobacterial community composition and dynamics in different aquatic systems. Our results open the door to future studies investigating picocyanobacterial diversity in aquatic systems. IMPORTANCE The molecular diversity of the aquatic picocyanobacterial community cannot be accurately described using only the available universal 16S rRNA gene primers that target the whole bacterial and archaeal community. We show that the hypervariable regions V5, V6, and V7 of the 16S rRNA gene are better suited to study the diversity, community structure, and dynamics of picocyanobacterial communities at a fine scale using Illumina MiSeq sequencing. Due to its variability, it allows reconstructing phylogenies featuring topologies comparable to those generated when using the complete 16S rRNA gene sequence. Further, we successfully designed a new set of primers flanking the V5 to V7 region whose specificity for picocyanobacterial genera was tested in silico and validated in several freshwater and marine aquatic communities. This work represents a step forward for understanding the diversity and ecology of aquatic picocyanobacteria and sets the path for future studies on picocyanobacterial diversity., (Copyright © 2019 American Society for Microbiology.)

The transformation of leucine incorporation rates to prokaryotic carbon production rates requires the use of either theoretical or empirically determined conversion factors. Empirical leucine-to-carbon conversion factors (eCFs) vary widely across environments, and little is known about their potential controlling factors. We conducted 10 surface seawater manipulation experiments across the world's oceans, where the growth of the natural prokaryotic assemblages was promoted by filtration (i.e., removal of grazers [F treatment]) or filtration combined with dilution (i.e., also relieving resource competition [FD treatment]). The impact of sunlight exposure was also evaluated in the FD treatments, and we did not find a significant effect on the eCFs. The eCFs varied from 0.09 to 1.47 kg C mol Leu(-1) and were significantly lower in the FD than in the F samples. Also, changes in bacterial community composition during the incubations, as assessed by automated ribosomal intergenic spacer analysis (ARISA), were more pronounced in the FD than in the F treatments, compared to unmanipulated controls. Thus, we discourage the common procedure of diluting samples (in addition to filtration) for eCF determination. The eCFs in the filtered treatment were negatively correlated with the initial chlorophyll a concentration, picocyanobacterial abundance (mostly Prochlorococcus), and the percentage of heterotrophic prokaryotes with high nucleic acid content (%HNA). The latter two variables explained 80% of the eCF variability in the F treatment, supporting the view that both Prochlorococcus and HNA prokaryotes incorporate leucine in substantial amounts, although this results in relatively low carbon production rates in the oligotrophic ocean., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

We analyzed the regional distribution of bulk heterotrophic prokaryotic activity (leucine incorporation) and selected single-cell parameters (cell viability and nucleic acid content) as parameters for microbial functioning, as well as bacterial and archaeal community structure in the epipelagic (0 to 200 m) and mesopelagic (200 to 1,000 m) subtropical Northeast Atlantic Ocean. We selectively sampled three contrasting regions covering a wide range of surface productivity and oceanographic properties within the same basin: (i) the eddy field south of the Canary Islands, (ii) the open-ocean NE Atlantic Subtropical Gyre, and (iii) the upwelling filament off Cape Blanc. In the epipelagic waters, a high regional variation in hydrographic parameters and bacterial community structure was detected, accompanied, however, by a low variability in microbial functioning. In contrast, mesopelagic microbial functioning was highly variable between the studied regions despite the homogeneous abiotic conditions found therein. More microbial functioning parameters indicated differences among the three regions within the mesopelagic (i.e., viability of cells, nucleic acid content, cell-specific heterotrophic activity, nanoflagellate abundance, prokaryote-to-nanoflagellate abundance ratio) than within the epipelagic (i.e., bulk activity, nucleic acid content, and nanoflagellate abundance) waters. Our results show that the mesopelagic realm in the Northeast Atlantic is, in terms of microbial activity, more heterogeneous than its epipelagic counterpart, probably linked to mesoscale hydrographical variations.

Growth is one of the basic attributes of any living organism. Surprisingly, the growth rates of marine bacterioplankton are only poorly known. Current data suggest that marine bacteria grow relatively slowly, having generation times of several days. However, some bacterial groups, such as the aerobic anoxygenic phototrophic (AAP) bacteria, have been shown to grow much faster. Two manipulation experiments, in which grazing, viruses, and resource competition were reduced, were conducted in the coastal Mediterranean Sea (Blanes Bay Microbial Observatory). The growth rates of AAP bacteria and of several important phylogenetic groups (the Bacteroidetes, the alphaproteobacterial groups Roseobacter and SAR11, and the Gammaproteobacteria group and its subgroups the Alteromonadaceae and the NOR5/OM60 clade) were calculated from changes in cell numbers in the manipulation treatments. In addition, we examined the role that top-down (mortality due to grazers and viruses) and bottom-up (resource availability) factors play in determining the growth rates of these groups. Manipulations resulted in an increase of the growth rates of all groups studied, but its extent differed largely among the individual treatments and among the different groups. Interestingly, higher growth rates were found for the AAP bacteria (up to 3.71 day⁻¹) and for the Alteromonadaceae (up to 5.44 day⁻¹), in spite of the fact that these bacterial groups represented only a very low percentage of the total prokaryotic community. In contrast, the SAR11 clade, which was the most abundant group, was the slower grower in all treatments. Our results show that, in general, the least abundant groups exhibited the highest rates, whereas the most abundant groups were those growing more slowly, indicating that some minor groups, such the AAP bacteria, very likely contribute much more to the recycling of organic matter in the ocean than what their abundances alone would predict.

An understanding of the distribution of colored dissolved organic matter (CDOM) in the oceans and its role in the global carbon cycle requires a better knowledge of the colored materials produced and consumed by marine phytoplankton and bacteria. In this work, we examined the net uptake and release of CDOM by a natural bacterial community growing on DOM derived from four phytoplankton species cultured under axenic conditions. Fluorescent humic-like substances exuded by phytoplankton (excitation/emission [Ex/Em] wavelength, 310 nm/392 nm; Coble's peak M) were utilized by bacteria in different proportions depending on the phytoplankton species of origin. Furthermore, bacteria produced humic-like substances that fluoresce at an Ex/Em wavelength of 340 nm/440 nm (Coble's peak C). Differences were also observed in the Ex/Em wavelengths of the protein-like materials (Coble's peak T) produced by phytoplankton and bacteria. The induced fluorescent emission of CDOM produced by prokaryotes was an order of magnitude higher than that of CDOM produced by eukaryotes. We have also examined the final compositions of the bacterial communities growing on the exudates, which differed markedly depending on the phytoplankton species of origin. Alteromonas and Roseobacter were dominant during all the incubations on Chaetoceros sp. and Prorocentrum minimum exudates, respectively. Alteromonas was the dominant group growing on Skeletonema costatum exudates during the exponential growth phase, but it was replaced by Roseobacter afterwards. On Micromonas pusilla exudates, Roseobacter was replaced by Bacteroidetes after the exponential growth phase. Our work shows that fluorescence excitation-emission matrices of CDOM can be a helpful tool for the identification of microbial sources of DOM in the marine environment, but further studies are necessary to explore the association of particular bacterial groups with specific fluorophores.

Since heterotrophic prokaryotes play an important biogeochemical role in aquatic ecosystems and have a high capacity to survive in extreme environments, easy-to-perform protocols that probe their physiological states and the effects of environmental variables on those states are highly desired. Some methodologies combine a general nucleic acid stain with a membrane integrity probe. We calibrated one of these, the nucleic acid double-staining (NADS) protocol (G. Grégori, S. Citterio, A. Ghiani, M. Labra, S. Sgorbati, S. Brown, and M. Denis, Appl. Environ. Microbiol. 67:4662-4670, 2001), determining the optimal stain concentrations in seawater and the response to conditions that generate prokaryote death (such as heat) and to conditions that are known to produce death in plankton, such as nutrient limitation or flagellate grazing. The protocol was validated by comparison to two methods used to detect viability: active respiration by 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and incorporation of tritiated leucine. We show that concentrations in the range of 5 to 20 microg ml(-1) of propidium iodide, simultaneous to a 10x concentration of Sybr green I, are best for detecting two separated populations of "live" (green cells) and "dead" (red cells) organisms. During exposure to heat and UVC, we observed that the number of live cells declined concurrently with that of actively respiring cells (CTC positive) and with total leucine incorporation. In seawater mesocosms, the NADS protocol allowed detection of bacterioplankton starvation-related death and flagellate predation. The protocol was also tested in deep profiles in the northwest Atlantic, demonstrating its potential for routine characterization of this fraction of the physiological diversity of marine heterotrophic prokaryotic plankton.

An annual seasonal cycle of composition of a bacterioplankton community in an oligotrophic coastal system was studied by denaturing gradient gel electrophoresis (DGGE) using five different primer sets. Analysis of DGGE fingerprints showed that primer set 357fGC-907rM grouped samples according to seasons. Additionally, we used the set of 16S rRNA genes archived in the RDPII database to check the percentage of perfect matches of each primer for the most abundant bacterial groups inhabiting coastal plankton communities. Overall, primer set 357fGC-907rM was the most suitable for the routine use of PCR-DGGE analyses in this environment.

We analyzed the contributions of different heterotrophic bacterial groups to the uptake of several low-molecular weight compounds during a seasonal cycle on the northwestern Mediterranean coast (Blanes Bay Microbial Observatory). The bacterial assemblage structure had been shown to change substantially year-round for this site, but whether changes in the activities of the different bacterial groups also occurred on the seasonal scale was unknown. Microautoradiography combined with catalyzed reporter deposition fluorescence in situ hybridization was used to analyze the patterns of glucose, amino acid, and ATP uptake by different bacterial groups. Gammaproteobacteria and Bacteroidetes were not very active in the uptake of glucose at any time of the year (<10% of cells were active) compared to Alphaproteobacteria (generally >20% of cells were active). Dissolved free amino acids were taken up considerably by Alphaproteobacteria and Gammaproteobacteria but not by Bacteroidetes. Relatively high percentages of cells of the three broad phylogenetic groups actively took up ATP, which could be related to the important phosphorous limitation of bacterial production during most of the year in Blanes Bay. The contribution of SAR11 to the uptake of the monomers was variable year-round, generally with fewer than 30% of the cells being active. By contrast, Roseobacter were highly overrepresented in the uptake of all the substrates throughout all the year, with more than 50% of cells being active in all the samples and for all substrates. Our results suggest that substantial changes in the activity of some phylogenetic groups of bacteria occur throughout the year.

We studied the effects of natural sunlight on heterotrophic marine bacterioplankton in short-term experiments. We used a single-cell level approach involving flow cytometry combined with physiological probes and microautoradiography to determine sunlight effects on the activity and integrity of the cells. After 4 h of sunlight exposure, most bacterial cells maintained membrane integrity and viability as assessed by the simultaneous staining with propidium iodide and SYBR green I. In contrast, a significant inhibition of heterotrophic bacterial activity was detected, measured by 5-cyano-2,3 ditolyl tetrazolium chloride reduction and leucine incorporation. We applied microautoradiography combined with catalyzed reporter deposition-fluorescence in situ hybridization to test the sensitivity of the different bacterial groups naturally occurring in the Northwestern Mediterranean to sunlight. Members of the Gammaproteobacteria and Bacteroidetes groups appeared to be highly resistant to solar radiation, with small changes in activity after exposure. On the contrary, Alphaproteobacteria bacteria were more sensitive to radiation as measured by the cell-specific incorporation of labeled amino acids, leucine, and ATP. Within Alphaproteobacteria, bacteria belonging to the Roseobacter group showed higher resistance than members of the SAR11 cluster. The activity of Roseobacter was stimulated by exposure to photosynthetic available radiation compared to the dark treatment. Our results suggest that UV radiation can significantly affect the in situ single-cell activity of bacterioplankton and that naturally dominating phylogenetic bacterial groups have different sensitivity to natural levels of incident solar radiation.

We studied the effects of nutrient availability and protistan grazing on bacterial dynamics and community composition (BCC) in different parts of the canyon-shaped Rímov reservoir (Czech Republic). The effects of protistan grazing on BCC were examined using a size fractionation approach. Water from the dam area with only bacteria (<0.8 microm), bacteria and heterotrophic nanoflagellates (<5 microm), or whole water were incubated in situ inside dialysis bags. Top-down or predator manipulations (size fractionation) were also combined with bottom-up or resource manipulations, i.e., transplantation of samples to the middle and upper inflow parts of the reservoir with increased phosphorus availability. Significant genotypic shifts in BCC occurred with transplantation as indicated by denaturing gradient gel electrophoresis. Using different probes for fluorescence in situ hybridization, we found that 10 to 50% of total bacteria were members of the phylogenetically small cluster of beta-proteobacteria (targeted with the probe R-BT065). These rod-shaped cells of very uniform size were vulnerable to predation but very fast growing and responded markedly to the different experimental manipulations. In all the grazer-free treatments, the members of the R-BT065 cluster showed the highest net growth rates of all studied bacterial groups. Moreover, their relative abundance was highly correlated with bacterial bulk parameters and proportions of bacteria with high nucleic acid (HNA) content. In contrast, increasing protistan bacterivory yielded lower proportions of R-BT065-positive and HNA bacteria substituted by increasing proportions of the class Actinobacteria, which profited from the enhanced protistan bacterivory.

We tested the sensitivity of coastal picophytoplankton exposed to natural sunlight in short-term experiments. Cell abundance and cell-specific chlorophyll fluorescence were significantly reduced in Prochlorococcus spp. but not in Synechococcus, whereas picoeukaryotes had an intermediate response. These results are the first direct evidence of a differential sensitivity to sunlight of these ubiquitous marine members of unicellular phytoplankton.

The effect of irradiance in the range of 400 to 700 nm or photosynthetically active radiation (PAR) on bacterial heterotrophic production estimated by the incorporation of 3H-leucine (referred to herein as Leu) was investigated in the northwestern Mediterranean Sea and in a coastal North Atlantic site, with Leu uptake rates ranging over 3 orders of magnitude. We performed in situ incubations under natural irradiance levels of Mediterranean samples taken from five depths around solar noon and compared them to incubations in the dark. In two of the three stations large differences were found between light and dark uptake rates for the surface most samples, with dark values being on average 133 and 109% higher than in situ ones. Data obtained in coastal North Atlantic waters confirmed that dark enclosure may increase Leu uptake rates more than threefold. To explain these differences, on-board experiments of Leu uptake versus irradiance were performed with Mediterranean samples from depths of 5 and 40 m. Incubations under a gradient of 12 to 1,731 micromol of photons m(-2) x s(-1) evidenced a significant increase in incorporation rates with increasing PAR in most of the experiments, with dark-incubated samples departing from this pattern. These results were not attributed to inhibition of Leu uptake in the light but to enhanced bacterial response when transferred to dark conditions. The ratio of dark to light uptake rates increased as dissolved inorganic nitrogen concentrations decreased, suggesting that bacterial nutrient deficiency was overcome by some process occurring only in the dark bottles.

Total bacterial abundances estimated with different epifluorescence microscopy methods (4',6-diamidino-2-phenylindole [DAPI], SYBR Green, and Live/Dead) and with flow cytometry (Syto13) showed good correspondence throughout two microcosm experiments with coastal Mediterranean water. In the Syto13-stained samples we could differentiate bacteria with apparent high DNA (HDNA) content and bacteria with apparent low DNA (LDNA) content. HDNA bacteria, "live" bacteria (determined as such with the Molecular Probes Live/Dead BacLight bacterial viability kit), and nucleoid-containing bacteria (NuCC) comprised similar fractions of the total bacterial community. Similarly, LDNA bacteria and "dead" bacteria (determined with the kit) comprised a similar fraction of the total bacterial community in one of the experiments. The rates of change of each type of bacteria during the microcosm experiments were also positively correlated between methods. In various experiments where predator pressure on bacteria had been reduced, we detected growth of the HDNA bacteria without concomitant growth of the LDNA bacteria, such that the percentage contribution of HDNA bacteria to total bacterial numbers (%HDNA) increased. This indicates that the HDNA bacteria are the dynamic members of the bacterial assemblage. Given how quickly and easily the numbers of HDNA and LDNA bacteria can be obtained, and given the similarity to the numbers of "live" cells and NuCC, the %HDNA is suggested as a reference value for the percentage of actively growing bacteria in marine planktonic environments.