Differential toxicity of bioorthogonal non-canonical amino acids (BONCAT) in Escherichia coli

Single-cell methods allow studying the activity of single bacterial cells, potentially shedding light on regulatory mechanisms involved in services like biochemical cycling. Bioorthogonal non-canonical amino acid tagging (BONCAT) is a promising method for studying bacterial activity in natural communities, using the methionine analogues L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG) to track protein production in single cells. Both AHA and HPG have been deemed non-toxic, but recent findings suggest that HPG affects bacterial metabolism. In this study we examined the effect of AHA and HPG on Escherichia coli with respect to acute toxicity and growth. E. coli exposed to 5.6–90 μM HPG showed no growth, and the growth rate was significantly reduced at >0.35 μM HPG, compared to the HPG-free control. In contrast, E. coli showed growth at concentrations up to 9 mM AHA. In assays where AHA or HPG were added during the exponential growth phase, the growth sustained but the growth rate was immediately reduced at the highest concentrations (90 μM HPG and 10 mM AHA). Prolonged incubations (20h) with apparently non-toxic concentrations suggest that the cells incorporating NCAAs fail to divide and do not contribute to the next generation resulting in the relative abundance of labelled cells to decrease over time. These results show that HPG and AHA have different impact on the growth of E. coli. Both concentration and incubation time affect the results and need to be considered when designing BONCAT experiments and evaluating results. Time course incubations are suggested as a possible way to obtain more reliable results.publishedVersio

Bioorthogonal Non-canonical Amino Acid Tagging Combined With Flow Cytometry for Determination of Activity in Aquatic Microorganisms

In this study, we have combined bioorthogonal non-canonical amino acid tagging (BONCAT) and flow cytometry (FCM) analysis, and we demonstrate the applicability of the method for marine prokaryotes. Enumeration of active marine bacteria was performed by combining the DNA stain SYBR Green with detection of protein production with BONCAT. After optimization of incubation condition and substrate concentration on monoculture of Escherichia coli, we applied and modified the method to natural marine samples. We found that between 10 and 30% of prokaryotes in natural communities were active. The method is replicable, fast, and allow high sample throughput when using FCM. We conclude that the combination of BONCAT and FCM is an alternative to current methods for quantifying active populations in aquatic environments.publishedVersio

Flow Cytometric Analysis of Bacterial Protein Synthesis: Monitoring Vitality After Water Treatment

Bacterial vitality after water disinfection treatment was investigated using bio-orthogonal non-canonical amino acid tagging (BONCAT) and flow cytometry (FCM). Protein synthesis activity and DNA integrity (BONCAT–SYBR Green) was monitored in Escherichia coli monocultures and in natural marine samples after UV irradiation (from 25 to 200 mJ/cm2) and heat treatment (from 15 to 45 min at 55°C). UV irradiation of E. coli caused DNA degradation followed by the decrease in protein synthesis within a period of 24 h. Heat treatment affected both DNA integrity and protein synthesis immediately, with an increased effect over time. Results from the BONCAT method were compared with results from well-known methods such as plate counts (focusing on growth) and LIVE/DEAD™ BacLight™ (focusing on membrane permeability). The methods differed somewhat with respect to vitality levels detected in bacteria after the treatments, but the results were complementary and revealed that cells maintained metabolic activity and membrane integrity despite loss of cell division. Similarly, analysis of protein synthesis in marine bacteria with BONCAT displayed residual activity despite inability to grow or reproduce. Background controls (time zero blanks) prepared using different fixatives (formaldehyde, isopropanol, and acetic acid) and several different bacterial strains revealed that the BONCAT protocol still resulted in labeled, i.e., apparently active, cells. The reason for this is unclear and needs further investigation to be understood. Our results show that BONCAT and FCM can detect, enumerate, and differentiate bacterial cells after physical water treatments such as UV irradiation and heating. The method is reliable to enumerate and explore vitality of single cells, and a great advantage with BONCAT is that all proteins synthesized within cells are analyzed, compared to assays targeting specific elements such as enzyme activity.publishedVersio

How microbial food web interactions shape the arctic ocean bacterial community revealed by size fractionation experiments

In the Arctic, seasonal changes are substantial, and as a result, the marine bacterial community composition and functions differ greatly between the dark winter and light-intensive summer. While light availability is, overall, the external driver of the seasonal changes, several internal biological interactions structure the bacterial community during shorter timescales. These include specific phytoplankton–bacteria associations, viral infections and other top-down controls. Here, we uncover these microbial interactions and their effects on the bacterial community composition during a full annual cycle by manipulating the microbial food web using size fractionation. The most profound community changes were detected during the spring, with ‘mutualistic phytoplankton’—Gammaproteobacteria interactions dominating in the pre-bloom phase and ‘substrate-dependent phytoplankton’—Flavobacteria interactions during blooming conditions. Bacterivores had an overall limited effect on the bacterial community composition most of the year. However, in the late summer, grazing was the main factor shaping the community composition and transferring carbon to higher trophic levels. Identifying these small-scale interactions improves our understanding of the Arctic marine microbial food web and its dynamics

Marine mimivirus relatives are probably large algal viruses

<p>Abstract</p> <p>Background</p> <p><it>Acanthamoeba polyphaga </it>mimivirus is the largest known ds-DNA virus and its 1.2 Mb-genome sequence has revealed many unique features. Mimivirus occupies an independent lineage among eukaryotic viruses and its known hosts include only species from the <it>Acanthamoeba </it>genus. The existence of mimivirus relatives was first suggested by the analysis of the Sargasso Sea metagenomic data.</p> <p>Results</p> <p>We now further demonstrate the presence of numerous "mimivirus-like" sequences using a larger marine metagenomic data set. We also show that the DNA polymerase sequences from three algal viruses (CeV01, PpV01, PoV01) infecting different marine algal species (<it>Chrysochromulina ericina</it>, <it>Phaeocystis pouchetii</it>, <it>Pyramimonas orientalis</it>) are very closely related to their homolog in mimivirus.</p> <p>Conclusion</p> <p>Our results suggest that the numerous mimivirus-related sequences identified in marine environments are likely to originate from diverse large DNA viruses infecting phytoplankton. Micro-algae thus constitute a new category of potential hosts in which to look for new species of <it>Mimiviridae</it>.</p

Synechococcus in the Atlantic Gateway to the Arctic Ocean

Increasing temperatures, with pronounced effects at high latitudes, have raised questions about potential changes in species composition, as well as possible increased importance of small-celled phytoplankton in marine systems. In this study, we mapped out one of the smallest and globally most widespread primary producers, the picocyanobacterium Synechococcus, within the Atlantic inflow to the Arctic Ocean. In contrast to the general understanding that Synechococcus is almost absent in polar oceans due to low temperatures, we encountered high abundances (up to 21,000 cells mL-1) at 79 °N, and documented their presence as far north as 82.5 °N. Covering an annual cycle in 2014, we found that during autumn and winter, Synechococcus was often more abundant than picoeukaryotes, which usually dominate the picophytoplankton communities in the Arctic. Synechococcus community composition shifted from a quite high genetic diversity during the spring bloom to a clear dominance of two specific operational taxonomic units (OTUs) in autumn and winter. We observed abundances higher than 1,000 cells mL-1 in water colder than 2 °C at seven distinct stations and size-fractionation experiments demonstrated a net growth of Synechococcus at 2 °C in the absence of nano-sized grazers at certain periods of the year. Phylogenetic analysis of petB sequences demonstrated that these high latitude Synechococcus group within the previously described cold-adapted clades I and IV, but also contributed to unveil novel genetic diversity, especially within clade I

The summer bacterial and archaeal community composition of the northern Barents Sea

Climate change related alterations in the Arctic have influences on the marine ecosystems, in particular on phytoplankton bloom dynamics. Since phytoplankton blooms are the main provider of carbon sources to the microbial loop, the bacterial and archaeal community are affected by the changes as well. Warmer water and less sea ice can lead to an earlier onset of phytoplankton blooms and consequently also to changes in the bacterial and archaeal community dynamics throughout Arctic summers. Here, we compared the bacterial and archaeal community composition during three summers (2018, 2019, and 2021) along a transect from the Barents Sea to the Arctic Ocean north of Svalbard. We used 16S rRNA gene sequencing to investigate changes in the communities in time and space. The main results showed that, Gammaproteobacteria (Nitrincolaceae), Bacteroidia (Polaribacter), and Alphaproteobacteria (SAR11 clade 1a members) dominated the bacterial and archaeal community in the surface waters but varied in abundance patterns between the years. The variations are potentially a result of different phytoplankton bloom stages and consequently differences in the availability of carbon sources. The distinctly different deep water communities were dominated by Candidatus Nitrosopumilus, Marinimicrobia, and members of the SAR324 clade in all years. The results indicate that changes in phytoplankton bloom dynamics can influence bacterial and archaeal community and thereby marine carbon cycling in surface waters, although direct links to the effects of global warming remain uncertain.publishedVersio

Phenology and Environmental Control of Phytoplankton Blooms in the Kong Håkon VII Hav in the Southern Ocean

Knowing the magnitude and timing of pelagic primary production is important for ecosystem and carbon sequestration studies, in addition to providing basic understanding of phytoplankton functioning. In this study we use data from an ecosystem cruise to Kong Håkon VII Hav, in the Atlantic sector of the Southern Ocean, in March 2019 and more than two decades of satellite-derived ocean color to study phytoplankton bloom phenology. During the cruise we observed phytoplankton blooms in different bloom phases. By correlating bloom phenology indices (i.e., bloom initiation and end) based on satellite remote sensing to the timing of changes in environmental conditions (i.e., sea ice, light, and mixed layer depth) we studied the environmental factors that seemingly drive phytoplankton blooms in the area. Our results show that blooms mainly take place in January and February, consistent with previous studies that include the area. Sea ice retreat controls the bloom initiation in particular along the coast and the western part of the study area, whereas bloom end is not primarily connected to sea ice advance. Light availability in general is not appearing to control the bloom termination, neither is nutrient availability based on the autumn cruise where we observed non-depleted macronutrient reservoirs in the surface. Instead, we surmise that zooplankton grazing plays a potentially large role to end the bloom, and thus controls its duration. The spatial correlation of the highest bloom magnitude with marked topographic features indicate that the interaction of ocean currents with sea floor topography enhances primary productivity in this area, probably by natural fertilization. Based on the bloom timing and magnitude patterns, we identified five different bloom regimes in the area. A more detailed understanding of the region will help to highlight areas with the highest relevance for the carbon cycle, the marine ecosystem and spatial management. With this gained understanding of bloom phenology, it will also be possible to study potential shifts in bloom timing and associated trophic mismatch caused by environmental changes.publishedVersio

Earlier sea-ice melt extends the oligotrophic summer period in the Barents Sea with low algal biomass and associated low vertical flux

The decrease in Arctic sea-ice extent and thickness as a result of global warming will impact the timing, duration, magnitude and composition of phytoplankton production with cascading effects on Arctic marine food-webs and biogeochemical cycles. Here, we elucidate the environmental drivers shaping the composition, abundance, biomass, trophic state and vertical flux of protists (unicellular eukaryotes), including phytoplankton, in the Barents Sea in late August 2018 and 2019. The two years were characterized by contrasting sea-ice conditions. In August 2018, the sea-ice edge had retreated well beyond the shelf break into the Nansen Basin (>82°N), while in 2019, extensive areas of the northwestern Barents Sea shelf (>79°N) were still ice-covered. These contrasting sea-ice conditions resulted in marked interannual differences in the pelagic protist community structure in this area. In August 2018, the protist community was in a post-bloom stage of seasonal succession characterized by oligotrophic surface waters and dominance of small-sized phytoplankton and heterotrophic protists (predominantly flagellates and ciliates) at most stations. In 2019, a higher contribution of autotrophs and large-celled phytoplankton, particularly diatoms, to total protist biomass compared to 2018 was reflected in higher chlorophyll a concentrations and suggested that the protist community was still in a late bloom stage at some stations. It is noteworthy that particularly diatoms contributed a considerably higher proportion to the protist biomass at the ice-covered stations in both years compared to the open-water stations. This pattern was also evident in the higher vertical protist biomass flux in 2019, dominated by dinoflagellates and diatoms, compared to 2018. Our results suggest that the predicted transition toward an ice-free Barents Sea will lengthen the oligotrophic summer period with low algal biomass and associated low vertical flux.publishedVersio

Seasonality of the Physical and Biogeochemical Hydrography in the Inflow to the Arctic Ocean Through Fram Strait

Eastern Fram Strait and the shelf slope region north of Svalbard is dominated by the advection of warm, salty and nutrient-rich Atlantic Water (AW). This oceanic heat contributes to keeping the area relatively free of ice. The last years have seen a dramatic decrease in regional sea ice extent, which is expected to drive large increases in pelagic primary production and thereby changes in marine ecology and nutrient cycling. In a concerted effort, we conducted five cruises to the area in winter, spring, summer and fall of 2014, in order to understand the physical and biogeochemical controls of carbon cycling, for the first time from a year-round point of view. We document (1) the offshore location of the wintertime front between salty AW and fresher Surface Water in the ocean surface, (2) thermal convection of Atlantic Water over the shelf slope, likely enhancing vertical nutrient fluxes, and (3) the importance of ice melt derived upper ocean stratification for the spring bloom timing. Our findings strongly confirm the hypothesis that this “Atlantification,” as it has been called, of the shelf slope area north of Svalbard resulting from the advection of AW alleviates both nutrient and light limitations at the same time, leading to increased pelagic primary productivity in this region