Specific rates of leucine incorporation by marine bacterioplantkon in the open Mediterranean Sea in summer using cell sorting

Abstract. Cell-specific leucine incorporation rates were determined in early summer across the open stratified Mediterranean Sea along vertical profiles from 0 to 200 m. During the period of our study, the bulk leucine incorporation rate was on average 5.0 ± 4.0 (n=31) pmol leu l−1 h−1. After 3H-radiolabeled leucine incorporation and SyBR Green I staining, populations were sorted using flow cytometry. Heterotrophic prokaryotes (Hprok) were divided in several clusters according to the cytometric properties of side scatter and green fluorescence of the cells: the low nucleic acid content cells (LNA) and the high nucleic acid content cells (HNA), with high size and low size (HNA-hs and HNA-ls, respectively). LNA cells represented 45 to 63% of the Hprok abundance between surface and 200 m, and significantly contributed to the bulk activity, from 17 to 55% all along the transect. The HNA/LNA ratio of cell-specific activities was on average 2.1 ± 0.7 (n=31). Among Hprok populations from surface samples (0 down to the deep chlorophyll depth, DCM), HNA-hs was mostly responsible for the leucine incorporation activity. Its cell-specific activity was up to 13.3 and 6.9-fold higher than that of HNA-ls and LNA, respectively, and it varied within a wide range of values (0.9–54.3×10−21 mol leu cell−1 h−1). At the opposite, ratios between the specific activities of the 3 populations tended to get closer to each other, below the DCM, implying a potentially higher homogeneity in activity of Hprok in the vicinity of nutriclines. Prochlorococcus cells were easily sorted near the DCM and displayed cell-specific activities equally high, sometimes higher than the HNA-hs group (2.5–55×10−21 mol leu cell−1 h−1). We then showed that all the sorted populations were key-players in leucine incorporation into proteins. The mixotrophic feature of certain photosynthetic prokaryotes and the non-negligible activity of LNA cells all over Mediterranean were reinforced.

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Does the High Nucleic Acid Content of Individual Bacterial Cells Allow Us To Discriminate between Active Cells and Inactive Cells in Aquatic Systems?

Applied and Environmental Microbiology ◽

10.1128/aem.67.4.1775-1782.2001 ◽

2001 ◽

Vol 67 (4) ◽

pp. 1775-1782 ◽

Cited By ~ 241

Author(s):

Philippe Lebaron ◽

Pierre Servais ◽

Helene Agogué ◽

Claude Courties ◽

Fabien Joux

Keyword(s):

Flow Cytometry ◽

Nucleic Acid ◽

Acid Content ◽

Fluorescent Dyes ◽

Specific Activity ◽

Aquatic Systems ◽

Nucleic Acid Content ◽

Leucine Incorporation ◽

Bacterial Cells ◽

Syto 13

ABSTRACT The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.

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Microbial Functioning and Community Structure Variability in the Mesopelagic and Epipelagic Waters of the Subtropical Northeast Atlantic Ocean

Applied and Environmental Microbiology ◽

10.1128/aem.07962-11 ◽

2012 ◽

Vol 78 (9) ◽

pp. 3309-3316 ◽

Cited By ~ 13

Author(s):

Federico Baltar ◽

Javier Arístegui ◽

Josep M. Gasol ◽

Gerhard J. Herndl

Keyword(s):

Community Structure ◽

Nucleic Acid ◽

Atlantic Ocean ◽

Acid Content ◽

Regional Distribution ◽

Nucleic Acid Content ◽

Leucine Incorporation ◽

Northeast Atlantic ◽

Wide Range ◽

Northeast Atlantic Ocean

ABSTRACTWe 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.

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Variations of bacterial-specific activity with cell size and nucleic acid content assessed by flow cytometry

Aquatic Microbial Ecology ◽

10.3354/ame028131 ◽

2002 ◽

Vol 28 ◽

pp. 131-140 ◽

Cited By ~ 104

Author(s):

P Lebaron ◽

P Servais ◽

AC Baudoux ◽

M Bourrain ◽

C Courties ◽

...

Keyword(s):

Flow Cytometry ◽

Nucleic Acid ◽

Cell Size ◽

Acid Content ◽

Specific Activity ◽

Nucleic Acid Content

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Geographic distribution pattern of low and high nucleic acid content bacteria on a river-catchment scale

Marine and Freshwater Research ◽

10.1071/mf16068 ◽

2017 ◽

Vol 68 (9) ◽

pp. 1618 ◽

Cited By ~ 3

Author(s):

Jie Liu ◽

Dan Ma ◽

Lili Ma ◽

Yuhao Song ◽

Guanghai Gao ◽

...

Keyword(s):

Nucleic Acid ◽

Acid Content ◽

Large River ◽

Nucleic Acid Content ◽

Catchment Scale ◽

Songhua River ◽

River Catchment ◽

Wide Range ◽

Biogeographical Pattern ◽

The Songhua River

Bacteria with low nucleic acid content (LNA) and high nucleic acid content (HNA) are widely distributed in aquatic environments. Most of the current understanding of these two subgroups is derived from studies in marine environments. In comparison, information on the spatial distribution of these two subgroups in freshwater environments is very limited. The present study analysed the biogeographical pattern of those two groups on a large-river scale (i.e. the Songhua River catchment, >1000km). The results showed that the concentrations of LNA and HNA bacteria were distributed over a wide range from 5.45×104 to 4.43×106cellsmL–1, and from 1.35×105 to 4.37×106cellsmL–1 respectively. The two groups have almost equal proportions in the Songhua River, with the average contribution of LNA bacteria reaching 47.0%. In comparison, the abundance of LNA bacteria in the mainstream was significantly higher than in the tributaries. The cytometric expressions (green fluorescence and side scatter) within LNA and HNA were strongly covaried, which implies that these two subgroups are intrinsically linked. Multivariate redundancy analysis indicated that both the abundance and cytometric characteristics of co-occurring LNA and HNA bacteria were regulated differently in the Songhua River. This suggests that LNA and HNA bacteria play different ecological roles in river ecosystems.

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Activity and Phylogenetic Diversity of Bacterial Cells with High and Low Nucleic Acid Content and Electron Transport System Activity in an Upwelling Ecosystem

Applied and Environmental Microbiology ◽

10.1128/aem.71.12.7737-7749.2005 ◽

2005 ◽

Vol 71 (12) ◽

pp. 7737-7749 ◽

Cited By ~ 79

Author(s):

K. Longnecker ◽

B. F. Sherr ◽

E. B. Sherr

Keyword(s):

Nucleic Acid ◽

Electron Transport ◽

Transport System ◽

Acid Content ◽

Phylogenetic Diversity ◽

Electron Transport System ◽

16S Rrna Genes ◽

Rrna Genes ◽

Nucleic Acid Content ◽

Leucine Incorporation

ABSTRACT We evaluated whether bacteria with higher cell-specific nucleic acid content (HNA) or an active electron transport system, i.e., positive for reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), were responsible for the bulk of bacterioplankton metabolic activity. We also examined whether the phylogenetic diversity of HNA and CTC-positive cells differed from the diversity of Bacteria with low nucleic acid content (LNA). Bacterial assemblages were sampled both in eutrophic shelf waters and in mesotrophic offshore waters in the Oregon coastal upwelling region. Cytometrically sorted HNA, LNA, and CTC-positive cells were assayed for their cell-specific [3H]leucine incorporation rates. Phylogenetic diversity in sorted non-radioactively labeled samples was assayed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Cell-specific rates of leucine incorporation of HNA and CTC-positive cells were on average only slightly greater than the cell-specific rates of LNA cells. HNA cells accounted for most bacterioplankton substrate incorporation due to high abundances, while the low abundances of CTC-positive cells resulted in only a small contribution by these cells to total bacterial activity. The proportion of the total bacterial leucine incorporation attributable to LNA cells was higher in offshore regions than in shelf waters. Sequence data obtained from DGGE bands showed broadly similar phylogenetic diversity across HNA, LNA, and CTC-positive cells, with between-sample and between-region variability in the distribution of phylotypes. Our results suggest that LNA bacteria are not substantially different from HNA bacteria in either cell-specific rates of substrate incorporation or phylogenetic composition and that they can be significant contributors to bacterial metabolism in the sea.

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Flow cytometric assessment of specific leucine incorporation in the open Mediterranean

Biogeosciences ◽

10.5194/bg-8-253-2011 ◽

2011 ◽

Vol 8 (2) ◽

pp. 253-265 ◽

Cited By ~ 19

Author(s):

A. Talarmin ◽

F. Van Wambeke ◽

P. Catala ◽

C. Courties ◽

P. Lebaron

Keyword(s):

Nucleic Acid ◽

Mediterranean Sea ◽

Early Summer ◽

Biogenic Elements ◽

Leucine Incorporation ◽

Surface Layers ◽

Flow Cytometric ◽

Dissolved Organic Compounds ◽

Heterotrophic Prokaryotes ◽

The Mediterranean

Abstract. The surface of the Mediterranean Sea is a low-phosphate-low-chlorophyll marine area where marine heterotrophic prokaryotes significantly contribute to the biogeochemical cycles of all biogenic elements such as carbon, notably through the mineralization of dissolved organic compounds. Cell-specific leucine incorporation rates were determined in early summer in the open stratified Mediterranean Sea. The bulk leucine incorporation rate was on average 5 ± 4 pmol leu l−1 h−1 (n=30). Cell-specific 3H-leucine incorporation rates were assayed using flow cytometry coupled to cell sorting. Heterotrophic prokaryotes (Hprok) were divided into cytometric groups according to their side scatter and green fluorescence properties: high nucleic acid containing cells (HNA) with high scatter (HNA-hs) and low scatter (HNA-ls) and low nucleic acid containing cells (LNA). Cell-specific leucine incorporation rates of these cytometric groups ranged from 2 to 54, 0.9 to 11, and 1 to 12 × 10-21 mol cell−1 h−1, respectively. LNA cells represented 45 to 63% of the Hprok abundance, and significantly contributed to the bulk leucine incorporation rates, from 12 to 43%. HNA/LNA ratios of cell-specific leucine incorporation were on average 2.0 ± 0.7 (n=30). In surface layers (from 0 m down to the deep chlorophyll depth, DCM), cell-specific rates of HNA-hs were elevated (7 and 13 times greater than LNA and HNA-ls, respectively). Nevertheless, on average HNA-hs (26%) and LNA (27%) equally contributed to the bulk leucine incorporation in these layers. Prochlorococcus cells were easily sorted near the DCM and displayed cell-specific leucine incorporation rates ranging from 3 to 55 × 10-21 mol leu cell−1 h−1, i.e. as high as HNA-hs'. These sorted groups could therefore be defined as key-players in the process of leucine incorporation into proteins. The mixotrophic features of certain photosynthetic prokaryotes and the high contribution of LNA cells to leucine incorporation within the microbial communities of the Mediterranean could be reinforced.

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Application of Ogur & Rosen's Method for the Determination of Nucleic Acid Content of Bacteria

Nippon Saikingaku Zasshi ◽

10.3412/jsb.12.125 ◽

1957 ◽

Vol 12 (2) ◽

pp. 125-129 ◽

Cited By ~ 3

Author(s):

Nobuyasu KAWASAKI ◽

Ichiro TAKI ◽

Chiaki WATANABE ◽

Kiyoshi MATOBA ◽

Mokichiro NISHIO ◽

...

Keyword(s):

Nucleic Acid ◽

Acid Content ◽

Nucleic Acid Content

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Using flow cytometry and light-induced fluorescence technique to characterizethe variability and characteristics of bioaerosols in springtime at Metro Atlanta, Georgia

10.5194/acp-2018-1073 ◽

2018 ◽

Author(s):

Arnaldo Negron ◽

Natasha DeLeon-Rodriguez ◽

Samantha M. Waters ◽

Luke D. Ziemba ◽

Bruce Anderson ◽

...

Keyword(s):

Flow Cytometry ◽

Nucleic Acid ◽

Acid Content ◽

Fungal Spores ◽

Epifluorescence Microscopy ◽

Nucleic Acid Content ◽

Rain Event ◽

Bacterial Cells ◽

Sampling System ◽

Spores And Pollen

Abstract. The abundance and speciation of primary biological aerosol particles (PBAP) is important for understanding their impacts on human health, cloud formation and ecosystems. Towards this, we have developed a protocol for quantifying PBAP collected from large volumes of air with a portable wet-walled cyclone bioaerosol sampler. A flow cytometry (FCM) protocol was then developed to quantify and characterize the PBAP populations from the sampler, which were confirmed against epifluorescence microscopy. The sampling system and FCM analysis were used to study PBAP in Atlanta, GA over a two-month period and showed clearly defined populations of DNA-containing particles: Low Nucleic Acid-content particles (bioLNA), High Nucleic Acid-content particles (HNA) being fungal spores and pollen. We find that daily-average springtime PBAP concentration (1 to 5 μm diameter) ranged between 1.4 × 104 and 1.1 × 105 m−3. The BioLNA population dominated PBAP during dry days (72 ± 18 %); HNA dominated the PBAP during humid days and following rain events, where HNA (e.g., wet-ejected fungal spores) comprised up to 92 % of the PBAP number. Concurrent measurements with a Wideband Integrated Bioaerosol Sensor (WIBS-4A) showed that FBAP and total FCM counts are similar; HNA (from FCM) significantly correlated with ABC type FBAP concentrations throughout the sampling period (and for the same particle size range, 1–5 μm diameter). However, the FCM bioLNA population, possibly containing bacterial cells, did not correlate to any FBAP type. The lack of correlation of any WIBS FBAP type with the bioLNA suggest bacterial cells may be more difficult to detect with autofluorescence than previously thought. Ιdentification of bacterial cells even in the FCM (bioLNA population) is challenging, given that the fluorescence level of stained cells at times may be comparable to that seen from abiotic particles. HNA and ABC displayed highest concentration on a humid and warm day after a rain event (4/14), suggesting that both populations correspond to wet-ejected fungal spores. Overall, information from both instruments combined reveals a highly dynamic airborne bioaerosol community over Atlanta, with a considerable presence of fungal spores during humid days, and a bioLNA population dominating bioaerosol community during dry days.

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