Depth Distribution of Bacterial Production in a Stratified Lake with an Anoxic Hypolimnion

The purpose of this study was to determine the depth distribution of bacterial biomass and production in a stratified lake and to test techniques to measure bacterial production in anaerobic waters. Bacterial abundance and incorporation of both [³H]thymidine and [³H]leucine into protein were highest in the metalimnion, at the depth at which oxygen first became unmeasurable. In contrast, [³H]thymidine incorporation into DNA was highest in the epilimnion. The ratios of incorporation into DNA/protein averaged 2.2, 0.49, and 0.95 for the epilimnion, metalimnion, and hypolimnion, respectively. Low incorporation into DNA was not due to artifacts associated with the DNA isolation procedure. Recovery of added [³H]DNA was about 90% in waters in which the portion of [³H]thymidine incorporation into DNA was about 40%. At least some obligate anaerobic bacteria were capable of assimilating thymidine since aeration of anaerobic hypolimnion waters substantially inhibited thymidine incorporation. The depth profile of bacterial production estimated from total thymidine and leucine incorporation and the frequency of dividing cells were all similar, with maximal rates in the metalimnion. However, estimates of bacterial production based on frequency of dividing cells and leucine incorporation were usually significantly higher than estimates based on thymidine incorporation (using conversion factors from the literature), especially in anaerobic hypolimnion waters. These data indicate that the thymidine approach must be examined carefully if it is to be applied to aquatic systems with low oxygen concentrations. Our results also indicate that the interface between the aerobic epilimnion and anaerobic hypolimnion is the site of intense bacterial mineralization and biomass production which deserves further study.     

Extracellular dissolved organic carbon released from phytoplankton as a source of carbon for heterotrophic bacteria in lakes of different humic content

The quantitative importance of photosynthetically produced dissolved organic carbon (PDOC) released from phytoplankton as a source of carbon for pelagic, heterotrophic bacteria was investigated in four temperate Swedish lakes, of which two had low (≈20 mg Pt 1⁻¹), and two moderately high (60–80 mg Pt 1⁻¹) humic content. The bacterial assimilation of PDOC was estimated with the ¹⁴C method, and the total production of the heterotrophic bacteria was estimated with the [3H]thymidine incorporation method. The release of PDOC from natural communities of phytoplankton was not restricted to periods of photosynthesis, but often continued during periods of darkness. Heterotrophic bacteria often assimilated the labile components of the PDOC at high rates (up to 73% of the released PDOC was assimilated during the incubation in our experiments). The contribution of PDOC to bacterial production exhibited large within-lake seasonal variations, but PDOC was at certain times, both in humic and non-humic lakes, a quantitatively very important carbon source for the heterotrophic bacteria. Under periods of comparatively low primary production, heterotrophic bacteria in humic lakes appear to utilize allochthonous, humic substances as a substrate.     

Bacterial metabolism in the River Danube: parameters influencing bacterial production

1. Microbial parameters were determined at five sampling sites in the River Danube up-and downstream of Vienna, Austria, twice monthly over an annual cycle. Bacterial production (BP) was estimated from thymidine and leucine incorporations; additionally, the effect of turbulence on BP and the conversion factors for converting incorporation rates into bacterial cell production were determined using the cumulative approach. 2. BP under turbulent conditions was not significantly different from that under stagnant conditions. For thymidine, a mean annual conversion factor of 3.2 ± 10¹⁸ cells mol^?1 thymidine incorporated was calculated. For leucine, the corresponding factor was 0.07 ± 10¹⁸ cells mol^?1 leucine. Average annual BP calculated by thymidine incorporation was significantly higher than BP calculated from leucine incorporation and ranged from 47.2 to 77.5 μg C 1-^?1 day^?1 depending on the tracer and the conversion factor used. 3. Bacterial growth rates ranged from 0.1 day^?1 during winter to 1.7 day^?1 in the summer. A strong correlation was found between temperature as well as chlorophyll a and bacterial growth when temperature was greater than 5 °C; a major spring phytoplankton bloom at a temperature below 5 °C did not increase BP. 4. Dissolved organic carbon (DOC) concentrations varied between 2 and 7.2 mg C 1-^?1 and comprised between 50 and 92% of the total organic carbon pool in the River Danube, Based on the DOC concentration and an assumed bacterial growth yield of 20% we calculated mean DOC turnover times of around 60 days in the winter and less than 8 days during the summer.     

MICROAUTORADIOGRAPHIC STUDIES OF THE MARINE PHYCOBIONTS RHIZOSOLENIA AND RICHELIA1

Microautoradiography was used to study photosynthetic and heterotrophic activities in cells of the diatom Rhizosolenia styliformis var. longispina containing the nitrogen-fixing, endophytic, blue-green alga Richelia intracellularis. In field studies with ¹⁴C-labeled bicarbonate, the blue-green alga appeared to be actively photosynthesizing but the diatom did not. Neither Richelia nor Rhizosolenia assimilated, ³H-labeled acetate, glucose, or an amino acid mixture. The diatom lacked cytoplasmic streaming and appeared, to be in a senescent condition. Implications of these observations for the nature of this algal association are discussed.     

Effects of Toxic Substances on Natural Bacterial Assemblages Determined by Means of [3H]Thymidine Incorporation

The effects of 3,5-dichlorophenol, 2,4-dinitrophenol, and potassium dichromate on natural bacterial assemblages were examined by means of [³H]thymidine incorporation into trichloroacetic acid-insoluble material. Results from a large number of coastal marine and freshwater samples suggest the following. (i) The effects of the three toxicants included reductions in the bacterial cell number as well as changes in rates of [³H]thymidine incorporation and in [³H]thymidine incorporation per cell. The concentrations that inhibited [³H]thymidine incorporation by 50% ranged from 3 to 11 mg liter⁻¹ for 3,5-dichlorophenol, 6 to 10 mg liter⁻¹ for 2,4-dinitrophenol, and 21 to 123 mg liter⁻¹ for potassium dichromate, with a tendency to higher values in bacterial assemblages from more eutrophic environments. (ii) The effects of 3,5-dichlorophenol and potassium dichromate determined by [³H]leucine incorporation into bacterial protein were similar or larger than those obtained from [³H]thymidine incorporation. (iii) Two to four hours of exposure to the toxicants was necessary before stable maximum effects were found in [³H]thymidine incorporation. (iv) Storage of natural environmental samples should be avoided, since tests with water stored for 1 to 3 days sometimes produced results different from results obtained from in situ tests. (v) The effects of 3,5-dichlorophenol, 2,4-dinitrophenol, and potassium dichromate on natural bacterial assemblages were relatively constant during periods with different growth rates in the assemblages, during various periods of the year, and between samples from freshwater and marine localities. With some precautions, [³H]thymidine incorporation can be used as a quick and sensitive method for determining the effects of toxicants on aquatic bacterial assemblages from natural environmental samples.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.     

The proton-sucrose symport

The heterotrophic tissues of the plant are dependent upon carbon and nitrogen import for normal growth and development. In general, oxidized forms of these essential elements are reductively assimilated in the leaf and, subsequently, sucrose and amino acids are transported to the heterotrophic cells in a process known as assimilate partitioning. In many plant species, a critical component of the assimilate partitioning pathway is the proton-sucrose symport. This active transport system couples sucrose translocation across the plasma membrane to the proton motive force generated by the H⁺-pumping ATPase. To date, the proton-sucrose symport is the only known system that can account for sucrose accumulation in the vascular tissue of the plant. This review focuses on recent advances describing the transport properties and bioenergetics of the proton-sucrose symport.     

Use of Microautoradiography Combined with Fluorescence In Situ Hybridization To Determine Dimethylsulfoniopropionate Incorporation by Marine Bacterioplankton Taxa

The fraction of planktonic heterotrophic bacteria capable of incorporating dissolved dimethylsulfoniopropionate (DMSP) and leucine was determined at two coastal sites by microautoradioagraphy (AU). In Gulf of Mexico seawater microcosm experiments, the proportion of prokaryotes that incorporated sulfur from [³⁵S]DMSP ranged between 27 and 51% of 4′,6-diamidino-2-phenylindole (DAPI)-positive cells, similar to or slightly lower than the proportion incorporating [³H]leucine. In the northwest Mediterranean coast, the proportion of cells incorporating sulfur from [³⁵S]DMSP increased from 5 to 42% from January to March, coinciding with the development of a phytoplankton bloom. At the same time, the proportion of cells incorporating [³H]leucine increased from 21 to 40%. The combination of AU and fluorescence in situ hybridization (FISH) revealed that the Roseobacter clade (α-proteobacteria) accounted for 13 to 43% of the microorganisms incorporating [³⁵S]DMSP at both sampling sites. Significant uptake of sulfur from DMSP was also found among members of the γ-proteobacteria and Cytophaga-Flavobacterium groups. Roseobacter and γ-proteobacteria exhibited the highest percentage of DAPI-positive cells incorporating ³⁵S from DMSP (around 50%). Altogether, the application of AU with [³⁵S]DMSP combined with FISH indicated that utilization of S from DMSP is a widespread feature among active marine bacteria, comparable to leucine utilization. These results point toward DMSP as an important substrate for a broad and diverse fraction of marine bacterioplankton.     

Estimating Bacterial Production in Marine Waters from the Simultaneous Incorporation of Thymidine and Leucine

We examined the simultaneous incorporation of [³H]thymidine and [¹⁴C]leucine to obtain two independent indices of bacterial production (DNA and protein syntheses) in a single incubation. Incorporation rates of leucine estimated by the dual-label method were generally higher than those obtained by the single-label method, but the differences were small (dual/single = 1.1 ± 0.2 [mean ± standard deviation]) and were probably due to the presence of labeled leucyl-tRNA in the cold trichloroacetic acid-insoluble fraction. There were no significant differences in thymidine incorporation between dual- and single-label incubations (dual/ single = 1.03 ± 0.13). Addition of the two substrates in relatively large amounts (25 nM) did not apparently increase bacterial activity during short incubations (<5 h). With the dual-label method we found that thymidine and leucine incorporation rates covaried over depth profiles of the Chesapeake Bay. Estimates of bacterial production based on thymidine and leucine differed by less than 25%. Although the need for appropriate conversion factors has not been eliminated, the dual-label approach can be used to examine the variation in bacterial production while ensuring that the observed variation in incorporation rates is due to real changes in bacterial production rather than changes in conversion factors or introduction of other artifacts.     

Thymidine and leucine incorporation in soil bacteria with different cell size

Thymidine and leucine incorporation into macromolecules of soil bacteria extracted by homogenization-centrifugation were measured after size-fractionation of the bacterial suspension through different sized filters (1.0, 0.8, 0.6, 0.4 m). The specific thymidine incorporation rate was highest for the unfiltered and 1.0 m filtered suspensions (approximately 10 × 10^–21 mol thymidine bacteria^–1 h^–1), but decreased to 1.39 × 10^–21 mol bacteria^–1 h^–1 for bacteria passing the 0.4 m filter. The proportion of culturable bacteria (percent colony forming units/acridine orange direct counts) also decreased with bacterial cell size from 5.0% for the unfiltered bacterial suspension to 0.8% in the 0.4 µm filtrate. A strong linear correlation (r ² = 0.995) was found between the specific thymidine incorporation rate and the proportion of culturable bacteria. Leucine incorporation gave similar results to the thymidine incorporation. No effects of cell size on the degree of isotope dilution or unspecific labeling of other macromolecules were found either for the thymidine or the leucine incorporation technique. These data indicate that small bacteria, although more numerous than larger ones, not only constitute a smaller proportion of the soil bacterial biomass than larger bacteria, but also contribute to a lesser degree to carbon transformations in soil.     

Studies on bacterial cell wall inhibitors III. 3-amino-3-deoxy-D-glucose,an inhibitor of bacterial cell wall synthesis

It was shown that 3-amino-3-deoxy-D-glucose, one of the constituents of the kanamycin molecule and a metabolite of Bacillus sp., inhibits the bacterial synthesis of cell wall. The antibiotic (100 μg/ml) significantly inhibits the growth of Straphylococcis aureus FDA 209P as well as the incorporation of DL-[¹⁴C]alanine into the acid-insoluble macromolecular fraction of its growing cells in the presence of chloramphenicol (100 μg/ml). In contrast, the antibiotic doed not affect the incorporation of [³H]thymidine, [³H]uridine and L-[¹⁴C]leucine. The other constituents of kanamycin, 6-amino-6-deoxy-D-glucose and deoxystreptamine do not inhibit the synthesis of bacterial cell wall peptidoglycan.     

Proliferative activity of gastric and duodenal endocrine cells in the rat

Summary The replicative activity and migration of gastrin, somatostatin and serotonin cells in rat stomach and doudenum was studied using combined immunocytochemistry and autoradiography after ³H thymidine pulse-labeling. Our results show that a small proportion of gastrin, somatostatin and serotonin immunoreactive cells displays proliferative activity. The overall labeling index ranged from 1.3% for gastric endocrine cells to 3.2% for duodenal endocrine cells.In a pulse chase experiment, labeling indices of immunoreactive cells were estimated at several time intervals after ³H thymidine administration. Significant differences in labeling index were not found. Migration of ³H thymidine labeled endocrine cells towards the luminal surface was not found in the stomach nor in the doudenum.It is concluded that 1) these endocrine cells have replicating activity; 2) the replicative activity of endocrine cells is higher in the duodenum than in the stomach; 3) the various cell types do not show significant differences in replicating activity and 4) endocrine cells did not seem to migrate to the luminal surface of the mucosa along with the other epithelial cells.     

Measurement of the incorporation rates of four amino acids into proteins for estimating bacterial production

In aquatic ecosystems, [³H]thymidine incorporation into bacterial DNA and [³H]leucine incorporation into proteins are usually used to estimate bacterial production. The incorporation rates of four amino acids (leucine, tyrosine, lysine, alanine) into proteins of bacteria were measured in parallel on natural freshwater samples from the basin of the river Meuse (Belgium). Comparison of the incorporation into proteins and into the total macromolecular fraction showed that these different amino acids were incorporated at more than 90% into proteins. From incorporation measurements at four subsaturated concentrations (range, 2–77 nm), the maximum incorporation rates were determined. Strong correlations (r > 0.91 for all the calculated correlations) were found between the maximum incorporation rates of the different tested amino acids over a range of two orders of magnitude of bacterial activity. Bacterial production estimates were calculated using theoretical and experimental conversion factors. The productions calculated from the incorporation rates of the four amino acids were in good concordance, especially when the experimental conversion factors were used (slope range, 0.91–1.11, and r > 0.91). This study suggests that the incorporation of various amino acids into proteins can be used to estimate bacterial production.     

Incorporation of 3H-Thymidine by Different Prokaryotic Groups in Relation to Temperature and Nutrients in a Lacustrine Ecosystem

The incorporation of [³H-methyl] thymidine (³H-TdR) by Eubacteria, bacterial groups (α- and β-Proteobacteria, Cytophaga–Flavobacter), and Archaea was measured according to temperature (7 and 17°C) and nutrient levels (nitrogen, phosphorus, and carbon) in a lacustrine system (Sep, France). Short-term incubation was performed using a combination of microautoradiography and fluorescent in situ hybridization. Irrespective of the temperatures and nutrients studied, all the major phylogenetic bacterial groups assimilated ³H-TdR, and in most of the treatments studied, the proportion of β-Proteobacteria taking up ³H-TdR was higher than those in the other bacterial groups. The proportion of Bacteria and different bacterial groups studied incorporating ³H-TdR were significantly increased, approximately 1.5-fold, by temperature except for α-Proteobacteria (7.6-fold). The nutrient effect was not the same for the different bacterial groups according to the temperatures studied. The proportions of α-Proteobacteria (at both temperatures) and Cytophaga–Flavobacter (at 7°C) taking up ³H-TdR were significantly decreased and increased by adding N and P, respectively. Also, adding N, P, and C increased and decreased the percentage of β-Proteobacteria incorporating ³H-TdR at 7 and 17°C, respectively. The archaeal community showed a similar proportion of active cells (i.e., ³H-TdR) to the bacterial community, and uptake of ³H-TdR by Archaea was significantly increased (P < 0.05) by both temperature and nutrients. Thus, the assimilation of ³H-TdR by bacterial groups and Archaea in lacustrine system is significantly controlled by both temperature and nutrients.     

Abundance and production of bacteria, and relationship to phytoplankton production, in a large tropical lake (Lake Tanganyika)

1. Abundance and bacterial production (BP) of heterotrophic bacteria (HBact) were measured in the north and south basins of Lake Tanganyika, East Africa, during seasonal sampling series between 2002 and 2007. The major objective of the study was to assess whether BP can supplement phytoplankton particulate primary production (particulate PP) in the pelagic waters, and whether BP and particulate PP are related in this large lake. HBact were enumerated in the 0–100 m surface layer by epifluorescence microscopy and flow cytometry; BP was quantified using ³H‐thymidine incorporation, usually in three mixolimnion layers (0–40, 40–60 and 60–100 m). 2. Flow cytometry allowed three subpopulations to be distinguished: low nucleic acid content bacteria (LNA), high nucleic acid content bacteria (HNA) and Synechococcus‐like picocyanobacteria (PCya). The proportion of HNA was on average 67% of total bacterial abundance, and tended to increase with depth. HBact abundance was between 1.2 × 10⁵ and 4.8 × 10⁶ cells mL⁻¹, and was maximal in the 0–40 m layer (i.e. roughly, the euphotic layer). Using a single conversion factor of 15 fg C cell⁻¹, estimated from biovolume measurements, average HBact biomass (integrated over a 100‐m water column depth) was 1.89 ± 1.05 g C m⁻². 3. Significant differences in BP appeared between seasons, especially in the south basin. The range of BP integrated over the 0–100 m layer was 93–735 mg C m⁻² day⁻¹, and overlapped with the range of particulate PP (150–1687 mg C m⁻² day⁻¹) measured in the same period of time at the same sites. 4. Depth‐integrated BP was significantly correlated to particulate PP and chlorophyll‐a, and BP in the euphotic layer was on average 25% of PP. 5. These results suggest that HBact contribute substantially to the particulate organic carbon available to consumers in Lake Tanganyika, and that BP may be sustained by phytoplankton‐derived organic carbon in the pelagic waters.     

Bacterial Standing Stock, Activity, and Carbon Production during Formation and Growth of Sea Ice in the Weddell Sea, Antarctica

Bacterial response to formation and growth of sea ice was investigated during autumn in the northeastern Weddell Sea. Changes in standing stock, activity, and carbon production of bacteria were determined in successive stages of ice development. During initial ice formation, concentrations of bacterial cells, in the order of 1 × 10⁸ to 3 × 10⁸ liter^-1, were not enhanced within the ice matrix. This suggests that physical enrichment of bacteria by ice crystals is not effective. Due to low concentrations of phytoplankton in the water column during freezing, incorporation of bacteria into newly formed ice via attachment to algal cells or aggregates was not recorded in this study. As soon as the ice had formed, the general metabolic activity of bacterial populations was strongly suppressed. Furthermore, the ratio of [³H]leucine incorporation into proteins to [³H]thymidine incorporation into DNA changed during ice growth. In thick pack ice, bacterial activity recovered and growth rates up to 0.6 day^-1 indicated actively dividing populations. However, biomass-specific utilization of organic compounds remained lower than in open water. Bacterial concentrations of up to 2.8 × 10⁹ cells liter^-1 along with considerably enlarged cell volumes accumulated within thick pack ice, suggesting reduced mortality rates of bacteria within the small brine pores. In the course of ice development, bacterial carbon production increased from about 0.01 to 0.4 μg of C liter^-1 h^-1. In thick ice, bacterial secondary production exceeded primary production of microalgae.     

Inhibition of Macrophage DNA Synthesis by Immunomodulators

Oil-induced guinea pig peritoneal exudate macrophages were found to incorporate actively [³H]thymidine without any tissue fluids such as conditioned medium, lymphokines or inflammatory tissue exudates. The [³H]thymidine incorporation was markedly suppressed by macrophage stimulants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS), while glucosamine incorporation was simultaneously increased by these stimulants. The degree of suppression of thymidine incorporation depended on the cell density, the concentrations of the stimulants, and sera or culture media used. The exposure of macrophages to MDP for 30 min was sufficient to cause significant suppression.     

Inhibition by specific monosaccharides of interleukin 2-induced thymocyte proliferation

When rat thymocytes are cultured for 3 days in serum-free medium and are stimulated to divide by interleukin 2 (IL 2), concanavalin A, or sodium periodate oxidation, addition to the medium of 10–25 mMd-ribose, 2-deoxy-d-ribose, or N-acetyl-d-galactosamine inhibits by 40% or more the incorporation of [³H]thymidine. d-ribose and lectin-free IL 2 generated from sodium periodate oxidation of rat spleen cells were used to study the characteristics of this inhibition and to test possible mechanisms of inhibition. Viability of thymocytes cultured with d-ribose is similar to that of cells cultured without this sugar. In order to be inhibitory, d-ribose has to be added to the cultures within the first 24 hr, and the inhibition can be prevented if the sugar is removed 18–24 hr after the start of culture. d-Ribose does not block the absorption of IL 2 by unstimulated rat thymocytes or by concanavalin A-generated thymic or splenic blast cells. When thymocytes are cultured with d-ribose for 24 hr, inactivated with mitomycin C, and then cultured for 3 days with fresh mitogenically stimulated cells, [³H]thymidine incorporation into the latter is not altered. This suggests that the sugar does not generate suppressor cells or suppressor supernates. d-Ribose does not appear to be a general metabolic inhibitor since [³H]leucine incorporation into thymocyte proteins and the release of [³H]leucine into medium after a 2-hr. [³H]leucine pulse are not altered by d-ribose. Trivial or artifactual effects (nonspecific cytotoxicity, changes in thymidine transport, or changes in isotonicity of the culture medium) cannot explain the inhibition. A hypothetical mechanism of inhibition is discussed.     

Adherence of Pseudomonas aeruginosa to inanimate polymers including biomaterials

Cells of Pseudomonas aeruginosa were adhered to polymethyl methacrylate, polyvinyl acetate, polyvinyl chloride, polyhydroxyethyl methacrylate, mixed-acrylic, silicone, and natural latex materials. Planktonic bacteria and bacteria that adhered to the test materials were compared for their uptake of either L-[3,4,5-³H] leucine or [methyl-³H] thymidine during growth in a minimal medium. Leucine incorporation was reduced and thymidine uptake was negligible in adherent bacteria for up to 8 h following primary attachment by which time cells in the planktonic state showed active uptake of both substrates. These reduced uptake periods correlated with lag phases of growth of adherent cells as determined with a sonication-release plate count procedure and analyses of adenosine triphosphate (ATP). The extent of the lag phase of the adherent populations was dependent on initial densities of adhered cells and the nature of the substratum. Received 02 December 1998/ Accepted in revised form 25 April 1999