Leucine Assimilation Research Articles

Diversity, activity, and abundance of benthic microbes in the Southeastern Mediterranean Sea.

Benthic microbes are key organisms in the oligotrophic Southeastern Mediterranean Sea (SEMS), yet their abundance, activity, and diversity in this rapidly changing basin are not fully understood. We investigated the prokaryotic and microfungal communities throughout years 2018-2020 at 27 stations (6-1900m water depths, down to 20cm below the sediment surface), in two transects with distinct downslope transport regimes, and along the eutrophic coastline. We estimated microbial abundance with flow cytometry, secondary production as leucine assimilation, and sequenced marker genes (the 16S rRNA and internal transcribed spacer) to assess diversity indices. The highest abundance (0.21 × 108cellsgr-1 sediment) was estimated at slope stations where we assumed substantial transport rates and found an accumulation of organic carbon. Secondary production was the highest nearshore (12±4ngCgr-1 h-1), and markedly declined offshore (0.5±0.9ngCgr-1 h-1). Populations of archaea (dominant Nitrososphaeria and Nanoarchaeia) and diverse bacteria were stable over three years, and taxonomic composition was dictated mainly by depth gradients. Saprotrophic and pathotrophic microfungi Ascomycota (70%±23%)andBasidiomycota (16%±18%)wereprevalent, whereas parasitic chytrids were abundant nearshore. Our results highlight the role of downslope transport, which enriched the typical deep-sea communities with anaerobic lineages, in shaping microbial populations near the continental slope.

Mesozooplankton taurine production and prokaryotic uptake in the northern Adriatic Sea

Dissolved free taurine, an important osmolyte in phytoplankton and metazoans, has been shown to be a significant carbon and energy source for prokaryotes in the North Atlantic throughout the water column. However, the extent of the coupling between taurine production and consumption over a seasonal cycle has not been examined yet. We determined taurine production by abundant crustacean zooplankton and its role as a carbon and energy source for several prokaryotic taxa in the northern Adriatic Sea over a seasonal cycle. Taurine concentrations were generally in the low nanomolar range, reaching a maximum of 22 nmol L−1 in fall during a Pseudonitzschia bloom and coinciding with the highest zooplankton taurine release rates. Taurine accounted for up to 5% of the carbon, 11% of the nitrogen, and up to 71% of the sulfur requirements of heterotrophic prokaryotes. Members of the Roseobacter clade, Alteromonas, Thaumarchaeota, and Euryarchaeota exhibited higher cell‐specific taurine assimilation rates than SAR11 cells. However, cell‐specific taurine and leucine assimilation were highly variable in all taxa, suggesting species and/or ecotype specific utilization patterns of taurine and dissolved free amino acids. Copepods were able to cover the bulk taurine requirements of the prokaryotic communities in fall and winter and partly in the spring–summer period. Overall, our study emphasizes the significance of taurine as a carbon and energy source for the prokaryotic community in the northern Adriatic Sea and the importance of crustacean zooplankton as a significant source of taurine and other organic compounds for the heterotrophic prokaryotic community.

Osmotrophic glucose and leucine assimilation and its impact on EPA and DHA content in algae.

The uptake of dissolved organic compounds, that is, osmotrophy, has been shown to be an efficient nutritional strategy for algae. However, this mode of nutrition may affect the biochemical composition, for example, the fatty acid (FA) contents, of algal cells. This study focused on the osmotrophic assimilation of glucose and leucine by selected seven algal strains belonging to chlorophytes, chrysophytes, cryptophytes, dinoflagellates and euglenoids. Our laboratory experiments with stable isotope labeling showed that osmotrophy occurred in four of the selected seven strains. However, only three of these produced long chain omega-3 FAs eicosapentaenoic acid (EPA; 20:5ω3) and docosahexaenoic acid (DHA; 22:6ω3). High glucose content (5 mg L−1) affected negatively on the total FAs of Mallomonas kalinae and the total omega-3 FAs of Cryptomonas sp. Further, glucose assimilation explained 35% (negative effect) and leucine assimilation 48% (positive effect) of the variation of EPA, DHA and the FAs related to their synthesis in Cryptomonas sp. Moderate glucose concentration (2 mg L−1) was found to enhance the growth of Cryptomonas ozolinii, whereas low leucine (20 µg L−1) enhanced the growth of M. kalinae. However, no systematic effect of osmotrophy on growth rates was detected. Our study shows that osmotrophic assimilation of algae is species and compound specific, and that the effects of the assimilated compounds on algal metabolism also varies depending on the species.

Differential Assimilation of Inorganic Carbon and Leucine by Prochlorococcus in the Oligotrophic North Pacific Subtropical Gyre.

The light effect on photoheterotrophic processes in Prochlorococcus, and primary and bacterial productivity in the oligotrophic North Pacific Subtropical Gyre was investigated using 14C-bicarbonate and 3H-leucine. Light and dark incubation experiments were conducted in situ throughout the euphotic zone (0–175 m) on nine expeditions to Station ALOHA over a 3-year period. Photosynthetrons were also used to elucidate rate responses in leucine and inorganic carbon assimilation as a function of light intensity. Taxonomic group and cell-specific rates were assessed using flow cytometric sorting. The light:dark assimilation rate ratios of leucine in the top 150 m were ∼7:1 for Prochlorococcus, whereas the light:dark ratios for the non-pigmented bacteria (NPB) were not significant different from 1:1. Prochlorococcus assimilated leucine in the dark at per cell rates similar to the NPB, with a contribution to the total community bacterial production, integrated over the euphotic zone, of approximately 20% in the dark and 60% in the light. Depth-resolved primary productivity and leucine incorporation showed that the ratio of Prochlorococcus leucine:primary production peaked at 100 m then declined steeply below the deep chlorophyll maximum (DCM). The photosynthetron experiments revealed that, for Prochlorococcus at the DCM, the saturating irradiance (Ek) for leucine incorporation was reached at approximately half the light intensity required for light saturation of 14C-bicarbonate assimilation. Additionally, high and low red fluorescing Prochlorococcus populations (HRF and LRF), co-occurring at the DCM, had similar Ek values for their respective substrates, however, maximum assimilation rates, for both leucine and inorganic carbon, were two times greater for HRF cells. Our results show that Prochlorococcus contributes significantly to bacterial production estimates using 3H-leucine, whether or not the incubations are conducted in the dark or light, and this should be considered when making assessments of bacterial production in marine environments where Prochlorococcus is present. Furthermore, Prochlorococcus primary productivity showed rate to light-flux patterns that were different from its light enhanced leucine incorporation. This decoupling from autotrophic growth may indicate a separate light stimulated mechanism for leucine acquisition.

Low microbial respiration of leucine at ambient oceanic concentration in the mixed layer of the central Atlantic Ocean

Bacterioplankton are the primary consumers of dissolved organic matter in the ocean, thus the quantification of bacterioplankton production (BP) is essential to our understanding of carbon cycling in the largest ecosystems on Earth. We compared BP, measured as the rate of 14C‐leucine or 3H‐leucine uptake at close to saturating concentration (20 nmol L−1), with ambient uptake measured from dilution bioassays. We hypothesized that saturation with leucine would lead to its respiration as a carbon source, thereby not truly representing ambient BP. Seawater was collected from the photic zone throughout the Atlantic Ocean. Respiration as a proportion of total consumption (uptake + respiration) of close to ambient (0.4 nmol L−1) and close to saturating (20 nmol L−1) 14C‐leucine concentrations were compared. Saturating 3H‐leucine additions overestimated ambient leucine uptake at low rates (200% ± 100% ambient) and underestimated uptake at high rates (90% + 20% ambient). The proportion of total leucine uptake that was respired was threefold higher for 20 nmol L−1 14C‐leucine additions than 0.4 nmol L−1 14C‐leucine additions (15% ± 8% and 5% ± 4%, respectively). Consequently, microbial efficiency of leucine assimilation—an indicator of bacterioplankton growth efficiency—was significantly higher and more stable at close to ambient 14C‐leucine additions than at saturating concentrations (95% ± 4% and 85% ± 8%, respectively). Thus, saturation of oligotrophic open Atlantic Ocean bacterioplankton with leucine, or other molecules indicative of microbial metabolism, leads to the measurement of a response to a nutrient addition, rather than an ambient measurement.

Structural evidence for the involvement of the residues Ser187 and Tyr422 in substrate recognition in the 3-methylcrotonyl-coenzyme A carboxylase from Pseudomonas aeruginosa

The enzyme 3-methylcrotonyl-CoA carboxylase from Pseudomonas aeruginosa (Pa-MCCase) is essential for the assimilation of leucine and acyclic monoterpenes. The structure of the Pa-MCCase was analysed by computational modelling to establish the molecular basis of substrate recognition. The active site is composed of two zones, which may play important roles in substrate recognition and catalysis. To further understand the interactions of the active site with the substrate, site-directed mutagenesis of the conserved residues S187 and R51 located in zone I, and F417, Y422 and G423 from zone II of the Pa-MCCase was carried out. The residue substitutions S187A and Y422D completely abolished the Pa-MCCase activity, whereas substitutions R51A, F417Y and G423A indicated that these residues are not essential. Interestingly, the residues R47, R51 and S187 form a well-defined pocket that may play important roles in substrate coupling to the Co-A motif. At zone one, mutation S187A was essential, but mutant R51A retained activity, suggesting that the R51 function could be relegated to neighbouring positive residues. Residue Y422 instead of contributing to substrate discrimination, it may participate in deprotonation of methyl group on MC-CoA, because it is located at adequate distances from the 3-methylcrotonyl-chain and carboxybiotin groups in the Pa-MCCase carboxylation site.

Effect of visible light on dimethylsulfoniopropionate assimilation and conversion to dimethylsulfide in the North Pacific Subtropical Gyre

The aim of the present study was to assess the effect of photosynthetically active radiation (PAR, 400 to 700 nm) on the utilization of dissolved dimethylsulfoniopropionate (DMSPd) by microbial communities in the oligotrophic North Pacific Subtropical Gyre, using 35 S-labeled substrate. Rates of DMSPd-sulfur (DMSPd-S) assimilation into macromolecules of microorganisms in surface mixed layer seawater were 39 to 78% higher in samples that were incubated in the light than in dark controls. There was no photoinhibition in the light range tested (up to 1200 µmol pho- tons m �2 s �1 ). Leucine assimilation, an index of bacterial protein production, was also photostimu- lated and was significantly correlated (r = 0.93) to DMSPd-S assimilation, suggesting that cells can respond to an increase in S requirements for protein synthesis by assimilating DMSPd-S. Light- driven changes in DMSPd-S assimilation were inversely and significantly correlated (r = �0.57) to changes in the corresponding dimethylsulfide (DMS) yield (i.e. DMS produced per DMSP con- sumed), in support of the hypothesis that DMS is produced only after cellular sulfur requirements are met. While light-driven changes in DMSPd-S assimilation appear to affect DMS yield, more than half of the DMSPd product pool remained unidentified; thus, in addition to assimilation, there may be other metabolic processes or products that can regulate DMS production.

Sunlight effects on the DMSP-sulfur and leucine assimilation activities of polar heterotrophic bacterioplankton

The influence of solar ultraviolet radiation and photosynthetically active radiation (PAR) on summertime marine bacterial uptake and assimilation of sulfur from radiolabeled dimethlysulfoniopropionate (35S-DMSP) was studied at four Arctic and two Antarctic stations. Incubations with 3H-leucine were also conducted for comparative purposes as a measurement of bacterial activity. Arctic waters were characterized by large numbers of colonial Phaeocystis pouchetii and higher DMSP concentrations than in the two diatom-dominated Antarctic samples. Exposure to full sunlight radiation (280–700 nm), and to a lesser extent to PAR + UVA (320–700 nm), generally decreased the bacterial assimilation of 3H-leucine with respect to darkness, and caused variable effects on 35S-DMSP assimilation. By using a single-cell approach involving microautoradiography we found high percentages of sulfur assimilating cells within the bacterial groups Gammaproteobacteria, Bacteroidetes, SAR11 and Roseobacter despite the varying DMSP concentrations between Arctic and Antarctic samples. The dominant SAR11 clade contributed 50–70% of the cells assimilating both substrates in the Arctic stations, whereas either Gammaproteobacteria or SAR11 were the largest contributors to 3H-leucine uptake in samples from the two Antarctic stations. Only one station was analyzed for single-cell 35S-DMSP assimilation in Antarctica, and Gammaproteobacteria were major contributors to its uptake, providing the first evidence for Antarctic bacteria actively taking up 35S-DMSP. PAR + UVA repeatedly increased the number of SAR11 cells assimilating 3H-leucine. This pattern also occurred with other 35S-DMSP assimilating groups, though not so consistently. Our results support a widespread capability of polar bacteria to assimilate DMSP-sulfur during the season of maximum DMSP concentrations, and show for the first time that all major polar taxa can be highly active at this assimilation under the appropriate circumstances. Our findings further confirm the role of sunlight as a modulator of heterotrophic carbon and sulfur fluxes in the surface ocean.

Effect of top-down control on cell-specific activity and diversity of active marine bacterioplankton

Mortality processes such as grazing and viral lysis can alter both phylogenetic diversity and cell-specific activity of bacterioplankton. We conducted experiments to examine the effect of re- duction in grazer or viral abundance on metabolically active bacterioplankton at a eutrophic shelf station and an oligotrophic basin station. Leucine assimilation was used as a proxy to characterize metabolically active bacterial cells. The phylogenetic affiliation of marine bacterioplankton assimilat- ing leucine was identified with fluorescence in situ hybridization and microautoradiography. Flow cytometric sorting of leucine-labeled cells quantified cell-specific activity of high nucleic acid (HNA) and low nucleic acid cells. The activity and diversity of the bacterial community at the oligotrophic station was more responsive to a reduction in mortality compared to the community at the eutrophic station. HNA cells at the oligotrophic station showed a 4-fold increase in cell-specific leucine incorpo- ration when the abundance of flagellates was reduced and an 8-fold increase when the abundance of flagellates, viruses, and bacterial cells was reduced. The abundance of active Alphaproteobacteria increased at the oligotrophic station when either grazer or viral abundance was reduced. Activity re- sponses were less striking at the eutrophic station, and the abundance of active Gammaproteobacte- ria showed a greater increase with reduced flagellates compared to the treatment with reduced fla- gellates, viruses, and bacterioplankton. Our results indicate both the presence and type of mortality process play a key role in structuring a bacterial community, and this effect varies in ecosystems of differing trophic state.

A small population of planktonic Flavobacteria with disproportionally high growth during the spring phytoplankton bloom in a prealpine lake

Bacterioplankton growth in temperate Lake Zurich (Switzerland) was studied during the spring phytoplankton bloom by in situ techniques and short-term dilution bioassays. A peak of chlorophyll a (Chl a) concentrations was followed by a rise of bacterial cell numbers and leucine assimilation rates, of the proportions of cells incorporating 5-bromo-2-deoxyuridine (BrdU), and of community net growth rates in dilution cultures. Incorporation of BrdU was low in Betaproteobacteria (2 +/- 1%), indicating that these bacteria did not incorporate the tracer. Pronounced growth of Betaproteobacteria in the enrichments was only observed after the decline of the phytoplankton bloom. An initial peak in the proportions of BrdU-positive Actinobacteria (30%) preceded a distinct rise of their cell numbers during the period of the Chl a maximum. Cytophaga-Flavobacteria (CF) changed little in numbers, but featured high proportions of BrdU-positive cells (28 +/- 12%). Moreover, CF represented > 90% of all newly formed cells in dilution cultures before and during the phytoplankton bloom. One phylogenetic lineage of cultivable Flavobacteria (FLAV2) represented a small (0.5-1%) but highly active population in lake plankton. The growth rates of FLAV2 in dilution cultures doubled during the period of the Chl a maximum, indicating stimulation by phytoplankton exudates. Thus, CF, and specifically Flavobacteria, appeared to be substantially more important for carbon transfer in Lake Zurich spring bacterioplankton than was suggested by their standing stocks. The high in situ growth potential of these bacteria might have been counterbalanced by top-down control.

Number and phylogenetic affiliation of bacteria assimilating dimethylsulfoniopropionate and leucine in the ice-covered coastal Arctic Ocean

The ability of bacteria to assimilate sulfur from dimethylsulfoniopropionate (DMSP) was examined in the western Arctic Ocean by combining microautoradiography and fluorescence in situ hybridization (FISH). Assimilation of leucine was also measured for comparative purposes since leucine is considered a universal substrate for bacteria, which use it for protein synthesis. Samples were collected at 3 m depth, through a hole in the ice, in the CASES (Canadian Arctic Shelf Ecosystem Study) overwintering station in Franklin Bay (eastern Beaufort Sea) in March and May 2004 to compare two contrasting situations: winter and early spring. FISH counts indicated that the bacterial assemblage consisted of α- (up to 60% of the EUB positive cells), β- (up to 10%) and γ-proteobacteria (around 20%), and Bacteroidetes (up to 60%). The β-proteobacteria were not active with any of the two substrates tested. The remaining groups were much less efficient at assimilating DMSP-sulfur (5% of the cells) than leucine (20–35%) both in winter and in spring. Only the Roseobacter group of α-proteobacteria showed a similar assimilation of both substrates.

Amino acid uptake from a probiotic milk in lactose intolerant subjects

This trial was designed to assess the effect of live probiotic consumption on leucine assimilation from fresh and pasteurised yoghurt in volunteers with different lactose digestibility. Thirty-three volunteers (mean age 32, s.d. 7 years) participated in this parallel single-blind study (16 of them with moderate lactose intolerance). Breath samples were taken before and at 15 min intervals over 3-h after the ingestion of fresh and pasteurised yoghurt extrinsically labelled with (1-(13)C)leucine. The 13C enrichment in breath was measured by isotopic rate mass spectrometry and mathematically converted to a percentage of assimilated leucine (100-%13C-dose in breath) and the assimilation kinetic constant (min(-1)). The 13C-leucine assimilation was statistically higher after the fresh yoghurt intake than after the pasteurised product intake (P=0.032) while the kinetic constant of assimilation was slower in intolerance status (P=0.014) although a product-related effect (P=0.445) was not found. In conclusion, fresh yoghurt intake resulted in higher short-term leucine assimilation, while lactose intolerance appears to negatively affect the assimilation rate of leucine from dairy products. These findings offer new insight on acute in vivo amino acid assimilation in the presence of probiotics and moderate lactose intolerance.

Light-Stimulated Bacterial Production and Amino Acid Assimilation by Cyanobacteria and Other Microbes in the North Atlantic Ocean

We examined the contribution of photoheterotrophic microbes--those capable of light-mediated assimilation of organic compounds--to bacterial production and amino acid assimilation along a transect from Florida to Iceland from 28 May to 9 July 2005. Bacterial production (leucine incorporation at a 20 nM final concentration) was on average 30% higher in light than in dark-incubated samples, but the effect varied greatly (3% to 60%). To further characterize this light effect, we examined the abundance of potential photoheterotrophs and measured their contribution to bacterial production and amino acid assimilation (0.5 nM addition) using flow cytometry. Prochlorococcus and Synechococcus were abundant in surface waters where light-dependent leucine incorporation was observed, whereas aerobic anoxygenic phototrophic bacteria were abundant but did not correlate with the light effect. The per-cell assimilation rates of Prochlorococcus and Synechococcus were comparable to or higher than those of other prokaryotes, especially in the light. Picoeukaryotes also took up leucine (20 nM) and other amino acids (0.5 nM), but rates normalized to biovolume were much lower than those of prokaryotes. Prochlorococcus was responsible for 80% of light-stimulated bacterial production and amino acid assimilation in surface waters south of the Azores, while Synechococcus accounted for on average 12% of total assimilation. However, nearly 40% of the light-stimulated leucine assimilation was not accounted for by these groups, suggesting that assimilation by other microbes is also affected by light. Our results clarify the contribution of cyanobacteria to photoheterotrophy and highlight the potential role of other photoheterotrophs in biomass production and dissolved-organic-matter assimilation.

Investigating microbial activities in compost using mushroom ( Agaricus bisporus) cultivation as an experimental system

Investigations into the dynamic nature of composting environments are necessary to understand and ultimately optimise the complex processes that occur. In this study, various parameters were measured to investigate physical, chemical and biological changes that occur in compost during the production of Agaricus bisporus. In addition to monitoring the compost samples during mushroom cultivation, uninoculated samples were maintained for comparative purposes. Principal components analysis of the variables measured showed a clear distinction between the thermophilic Phase I composts, uninoculated Phase II composts and mushroom inoculated composts. Leucine assimilation, a novel technique to composting environments, is presented as suitable method for assessing microbial activity in such systems. Strict agreement between leucine assimilation and fluorescein diacetate (FDA) hydrolysis, a rarely used technique in composting environments, was not observed, suggesting that neither should be used as a sole measure of microbial activity in compost. Association of FDA hydrolysis with the culturable heterotrophic count suggests that FDA hydrolysis may indicate bacterial as opposed to total microbial activity.

Contribution of major bacterial groups to bacterial biomass production along a salinity gradient in the South China Sea

Bacterial abundance and assimilation of 3 H-leucine were examined using a combination of microautoradiography and fluorescent in situ hybridization (Micro-FISH) to determine the bacterioplankton community structure and estimate the contribution of major bacterial groups to total bacterial biomass production (leucine incorporation) from the Pearl River mouth to the open water area in the South China Sea. Alpha-, beta-, gamma-proteobacteria and the Cytophaga-Flavo- bacterium cluster differed substantially in their relative abundance and contribution to leucine assim- ilation along the salinity gradient. The contribution of major bacterial groups to leucine assimilation was closely associated with their relative abundance in the communities. Alpha-proteobacteria were the dominant group in the high-salinity water in terms of abundance and 3 H-leucine assimilation, whereas beta-proteobacteria were more important in fresh water. At all stations, gamma-proteobac- teria were a minor component, but the Cytophaga-Flavobacterium cluster was always a significant component in both marine and freshwater systems. The relative abundance of bacterial groups explained the variation in 3 H-leucine assimilation by the major bacterial groups to a great extent. At the singlebacterial group level, a statistically significant correlation between abundance and leucine- uptake activity was observed for beta-proteobacteria, suggesting that the relative abundance of beta- proteobacteria in bacterioplankton communities was controlled by growth-related processes. In general, the numerical dominance of the major phylogenetic groups and their contribution to total bacterial biomass production varied consistently along the salinity gradient in the South China Sea.

The effects of sewage-treatment-works effluent on epilithic bacterial and algal communities of three streams in northern england

The responses of epilithic bacterial and algal communities to sewage-treatment-works (STW) effluent were studied in three streams in North Yorkshire, England, using both conventional microbiological techniques and the techniques of molecular genomics. Cod Beck, Thornton Beck and the River Wiske, were visited in May–June 2000 and January 2001 and the epilithic communities on submerged stones were sampled to determine chlorophyll-a, leucine assimilation, bacterial abundance, identity and abundance of microalgae, and epilithon dry and organic weights. Additionally, DNA from the epilithon of stones, collected in March–April and November 2000, was extracted, and samples were amplified using universal primers appropriate, respectively, for bacteria [Muyzer et al., 1993. Applied and Environmental Microbiology 59: 695–700] and microscopic phototrophs [Nubel et al., 1997. Applied and Environmental Microbiology 63: 3327–3332] followed by DGGE (Denaturing Gradient Gel Electrophoresis). Differences in algal community composition and abundance were greater between streams than between sites upstream and downstream of the STW outfalls. DGGE banding profiles of bacterial communities revealed grouping according to stream, and not the formation of communities characteristic of downstream sites; seasonal variation was also evident. Thus the discharge of STW effluent to the streams did not bring about the development of communities that are characteristically associated with organic pollution. Changes in composition and structure of bacterial and micro-algal epilithic communities were detected, but the relatively high quality effluents had only mild effects, not altering the environments sufficiently to override the natural differences between the three streams.

The response of epilithic bacteria to different metals regime in two upland streams: assessed by conventional microbiological methods and PCR-DGGE

The epilithic bacterial community in the zinc-contaminated River West Allen, northeast England, was compared to that in the less contaminated River East Allen. Bacterial abundance (DAPI counts) and activity (leucine assimilation rate) were similar in the two streams, suggesting the possibility that a tolerant community had developed in the West Allen. Despite the similarity in abundance and activity, molecular genomic methods (PCR-DGGE) showed that the epilithic bacterial communities were consistently different between the two streams, and that bacterial diversity was greater in the zinc-contaminated West Allen. Sequencing of DGGE bands indicated that α-Proteobacteria and Nitrospirae, previously associated with metal-rich environments, were present in the West Allen. This may support the suggestion that a metal-tolerant bacterial community had developed at the zinc-contaminated site.

Biomass production and assimilation of dissolved organic matter by SAR11 bacteria in the Northwest Atlantic Ocean.

Members of the SAR11 clade often dominate the composition of marine microbial communities, yet their contribution to biomass production and the flux of dissolved organic matter (DOM) is unclear. In addition, little is known about the specific components of the DOM pool utilized by SAR11 bacteria. To better understand the role of SAR11 bacteria in the flux of DOM, we examined the assimilation of leucine (a measure of biomass production), as well as free amino acids, protein, and glucose, by SAR11 bacteria in the Northwest Atlantic Ocean. We found that when SAR11 bacteria were >25% of total prokaryotes, they accounted for about 30 to 50% of leucine incorporation, suggesting that SAR11 bacteria were major contributors to bacterial biomass production and the DOM flux. Specific growth rates of SAR11 bacteria either equaled or exceeded growth rates for the total prokaryotic community. In addition, SAR11 bacteria were typically responsible for a greater portion of amino acid assimilation (34 to 61%) and glucose assimilation (45 to 57%) than of protein assimilation (< or = 34%). These data suggest that SAR11 bacteria do not utilize various components of the DOM pool equally and may be more important to the flux of low-molecular-weight monomers than to that of high-molecular-weight polymers.

Quantifying nutrient production by the microbial symbionts in an aphid.

The symbiotic bacteria Buchnera sp. provide aphids with essential amino acids, nutrients in short supply in the aphid diet of plant phloem sap. The contribution of Buchnera-derived amino acids to net protein growth of the aphid Aphis fabae was quantified from the protein growth of aphids reared on chemically defined diets lacking individual amino acids. The amino acid production rates varied among the nine essential amino acids over the range 8-156 pmol microg(-1)protein day(-1) (for tryptophan and leucine, respectively), equivalent to 0.02-0.33 fmol Buchnera(-1)day(-1). In a complementary metabolic analysis, the aphids incorporated radioactivity from dietary [(14)C]glutamic acid into the essential amino acids isoleucine, lysine and threonine. Incorporation into isoleucine was significantly elevated by the omission of dietary isoleucine, indicating that dietary supply may affect the biosynthetic rates of certain amino acids by Buchnera. Aphids experimentally deprived of Buchnera did not synthesize essential amino acids from dietary glutamic acid. The mortality of aposymbionts was high over 7 days on the phenylalanine-free diet, and their assimilation of dietary leucine was depressed on the complete diet, suggesting that both the absence of bacteria-derived amino acids and the low rates of assimilation of certain dietary amino acids may contribute to the poor growth of these insects.

Downstream change in leucine aminopeptidase activity and leucine assimilation by epilithic microbiota along the River Swale, northern England

Parallel determinations of epilithic extracellular leucine aminopeptidase activity and leucine assimilation were made at five sites along 112 km of the River Swale and also in two tributaries, the River Wiske and Cod Beck. Epilithic leucine aminopeptidase activity along the Swale increased with distance downstream; this increase was gradual, rather than stepwise in response to specific sewage-works outfalls. Epilithic leucine assimilation, in contrast, did not consistently increase along the river. Epilithic leucine aminopeptidase activity and leucine assimilation were both potentially controlled by epilithic microbial variables (bacterial abundance and chlorophyll a) while leucine aminopeptidase activity was also strongly related to water-quality variables, especially temperature, pH and conductivity. Epilithic leucine aminopeptidase activity and leucine assimilation were coupled, but the magnitude of aminopeptidase activity was always substantially greater than that of leucine assimilation. Arguments are presented, however, which suggest that this did not necessarily indicate the constant availability of excess leucine, and by inference amino-acid nitrogen, to epilithic bacteria. Values of epilithic leucine aminopeptidase activity and leucine assimilation, expressed relative to rates in overlying water, suggested that most activity and assimilation was epilithic rather than planktonic, although the planktonic contribution was proportionately greater at the deeper, more downstream, sites. In the tributaries, River Wiske and Cod Beck, values of epilithic leucine aminopeptidase activity and epilithic microbial abundance, as well as those of many water-quality variables, resembled values in the middle and lower Swale. Thus, these tributaries were essentially lowland, enriched watercourses being very different from the headstreams of the main river.