Anoxygenic phototroph of the Chloroflexota uses a type I reaction centre.

Scientific exploration of phototrophic bacteria over nearly 200 years has revealed large phylogenetic gaps between known phototrophic groups that limit understanding of how phototrophy evolved and diversified1,2. Here, through Boreal Shield lake water incubations, we cultivated an anoxygenic phototrophic bacterium from a previously unknown order within the Chloroflexota phylum that represents a highly novel transition form in the evolution of photosynthesis. Unlike all other known phototrophs, this bacterium uses a type I reaction centre (RCI) for light energy conversion yet belongs to the same bacterial phylum as organisms that use a type II reaction centre (RCII) for phototrophy. Using physiological, phylogenomic and environmental metatranscriptomic data, we demonstrate active RCI-utilizing metabolism by the strain alongside usage of chlorosomes3 and bacteriochlorophylls4 related to those of RCII-utilizing Chloroflexota members. Despite using different reaction centres, our phylogenomic data provide strong evidence that RCI-utilizing and RCII-utilizing Chloroflexia members inherited phototrophy from a most recent common phototrophic ancestor. The Chloroflexota phylum preserves an evolutionary record of the use of contrasting phototrophic modes among genetically related bacteria, giving new context for exploring the diversification of phototrophy on Earth.

Anoxygenic photosynthesis and iron-sulfur metabolic potential of Chlorobia populations from seasonally anoxic Boreal Shield lakes.

Aquatic environments with high levels of dissolved ferrous iron and low levels of sulfate serve as an important systems for exploring biogeochemical processes relevant to the early Earth. Boreal Shield lakes, which number in the tens of millions globally, commonly develop seasonally anoxic waters that become iron rich and sulfate poor, yet the iron-sulfur microbiology of these systems has been poorly examined. Here we use genome-resolved metagenomics and enrichment cultivation to explore the metabolic diversity and ecology of anoxygenic photosynthesis and iron/sulfur cycling in the anoxic water columns of three Boreal Shield lakes. We recovered four high-completeness and low-contamination draft genome bins assigned to the class Chlorobia (formerly phylum Chlorobi) from environmental metagenome data and enriched two novel sulfide-oxidizing species, also from the Chlorobia. The sequenced genomes of both enriched species, including the novel "Candidatus Chlorobium canadense", encoded the cyc2 gene that is associated with photoferrotrophy among cultured Chlorobia members, along with genes for phototrophic sulfide oxidation. One environmental genome bin also encoded cyc2. Despite the presence of cyc2 in the corresponding draft genome, we were unable to induce photoferrotrophy in "Ca. Chlorobium canadense". Genomic potential for phototrophic sulfide oxidation was more commonly detected than cyc2 among environmental genome bins of Chlorobia, and metagenome and cultivation data suggested the potential for cryptic sulfur cycling to fuel sulfide-based growth. Overall, our results provide an important basis for further probing the functional role of cyc2 and indicate that anoxygenic photoautotrophs in Boreal Shield lakes could have underexplored photophysiology pertinent to understanding Earth's early microbial communities.

Millions of Boreal Shield Lakes can be used to Probe Archaean Ocean Biogeochemistry.

Life originated in Archaean oceans, almost 4 billion years ago, in the absence of oxygen and the presence of high dissolved iron concentrations. Early Earth oxidation is marked globally by extensive banded iron formations but the contributing processes and timing remain controversial. Very few aquatic habitats have been discovered that match key physico-chemical parameters of the early Archaean Ocean. All previous whole ecosystem Archaean analogue studies have been confined to rare, low sulfur, and permanently stratified lakes. Here we provide first evidence that millions of Boreal Shield lakes with natural anoxia offer the opportunity to constrain biogeochemical and microbiological aspects of early Archaean life. Specifically, we combined novel isotopic signatures and nucleic acid sequence data to examine processes in the anoxic zone of stratified boreal lakes that are naturally low in sulfur and rich in ferrous iron, hallmark characteristics predicted for the Archaean Ocean. Anoxygenic photosynthesis was prominent in total water column biogeochemistry, marked by distinctive patterns in natural abundance isotopes of carbon, nitrogen, and iron. These processes are robust, returning reproducibly after water column re-oxygenation following lake turnover. Evidence of coupled iron oxidation, iron reduction, and methane oxidation affect current paradigms of both early Earth and modern aquatic ecosystems.

Maturation of murine natural killer precursor cells in the absence of exogenous cytokines requires contact with bone marrow stroma.

To test whether differentiation of natural killer (NK) precursor cells in unsupplemented long-term bone marrow culture requires contact with stromal cells, bone marrow cells (BMC) that were depleted of NK 1.1+ and Thy-1+ cells were cultured either directly on established, irradiated bone marrow stroma or in cell culture inserts separated from the stroma by a 0.45-micron microporous membrane. No exogenous cytokines were added. Depleted BMC produced NK 1.1+ nonlytic cells by day 7 and lytic NK 1.1+ cells by day 14 only when grown directly on the stroma. NK precursor cells survived in the inserts, however, and could respond to recombinant interleukin-2 in secondary culture. Thus, stromal cell-dependent proliferation and differentiation of lytic NK 1.1+ cells from their nonlytic NK 1.1- precursors requires direct interaction between stromal cells and NK precursors.

Hamster monoclonal antibodies to murine natural killer cells.

To further the study of natural killer (NK) cells we have produced hamster monoclonal antibodies (MAbs) with reactivities to mouse NK cells. MAbs 4A2 and 3C2 were obtained by fusing spleen cells from Syrian hamsters immunized with IL-2-activated NK cells with Fox-NY myeloma cells. 4A2 antigen was expressed by bone marrow (BM)-derived IL-2-responsive NK cell precursors, by mature NK cells of the BM, and by a highly lytic subset of splenic NK cells, in addition to IL-2-activated NK cells. 3C2 antigen was also expressed by BM-derived NK cell precursors, by mature NK cells in the BM, at low levels by splenic NK cells, and at high levels by IL-2-activated NK cells. These MAbs are likely to provide useful reagents for the study of the life history and functional significance of NK cells.

Identification of cholate as a shared substrate for the unidirectional efflux systems for methotrexate in L1210 mouse cells.

The bidirectional transport properties of cholate have been examined in leukemic L1210 mouse cells and compared with the transport of methotrexate. The cell entry of [3H]cholate was Na(+)-independent, linear with increasing concentrations of substrate, enhanced by decreasing pH, and uneffected by excess unlabeled cholate or by various anion-transport inhibitors and hence had the characteristics of passive diffusion or a pH-dependent mediated process with a high Kt for cholate. The efflux of [3H]cholate, however, could be attributed to carrier-mediated and energy-dependent transport. Efflux was rapid (t1/2 = 1.5 min) and could be increased with glucose and decreased with metabolic inhibitors, and it was inhibited by various compounds including bromosulfophthalein, probenecid, prostaglandin A1, reserpine, verapamil, quinidine, diamide, 1-methyl-3-isobutylxanthine and vincristine. The most potent inhibitor was prostaglandin A1, which reduced efflux by 50% at a concentration of 0.10 microM. Half-maximal inhibition by vincristine occurred at 4.8 microM. The maximum extent of inhibition with most of the inhibitors was 95%, although a lower value was observed with bromosulfophthalein (85%). When cholate efflux was compared with the efflux of methotrexate, both processes responded similarly to changes in the metabolic state of the cell. Moreover, the various inhibitors of cholate efflux also inhibited the efflux of methotrexate and the same concentration of each inhibitor was required for half-maximal inhibition of both processes. The efflux of folate and urate also proceeded via outwardly directed, unidirectional processes which were sensitive to bromosulfophthalein and probenecid. The results suggest that L1210 cells have the capacity for the unidirectional extrusion of cholate, methotrexate and probably other large, structurally dissimilar organic anions and that this efflux occurs via two or more very similar transport systems with a broad anion specificity. The function of an organic anion efflux system in vivo may be to facilitate the extrusion of cytotoxic metabolic anions which are too large to exit via the general anion-exchange carrier of these cells. Similarities in inhibitor specificity were also apparent between unidirectional anion efflux in L1210 cells and the drug efflux pump which is over-produced in cells with multidrug resistance.

Identification of the bromosulfophthalein-sensitive efflux route for methotrexate as the site of action of vincristine in the vincristine-dependent enhancement of methotrexate uptake in L1210 cells.

The mechanism by which vincristine enhances the uptake of methotrexate in leukemic L1210 mouse cells has been investigated. Methotrexate uptake after 30 min at 37 degrees C increased 44% relative to untreated controls in cells suspended in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid/bicarbonate-buffered saline medium containing 20 microM vincristine. This stimulation was half-maximal at a vincristine concentration of 4 microM. An enhancement of methotrexate uptake by vincristine was also observed in the presence of glucose but a reversal could be achieved by prior treatment of the cells with taxol, a drug which prevents microtubule disassembly by vincristine. When the effect of vincristine was determined on the individual influx and efflux components for methotrexate, the increased uptake of methotrexate correlated with the inhibition of the unidirectional efflux route for methotrexate that is sensitive to bromosulfophthalein. Inhibition of this route was half-maximal at 3 microM vincristine and it exceeded 90% at high concentrations of the inhibitor. Transport via the bidirectional exchange carrier for methotrexate was not affected by vincristine, while the unidirectional efflux route sensitive to probenecid was inhibited by vincristine but only at concentrations 10-fold higher than required to inhibit the bromosulfophthalein-sensitive route. Vincristine did not increase methotrexate uptake in CCRF-CEM lymphoblasts, a human cell line which contains much lower levels of bromosulfophthalein-sensitive efflux route for methotrexate. Concentrations of vincristine which inhibited the bromosulfophthalein-sensitive efflux route of L1210 cells by 80-90% had little or no effect on the intracellular pH or on intracellular levels of ATP, GTP, cyclic AMP, K+, or oxidized glutathione. A significant increase was observed in the cellular uptake of tetraphenylphosphonium ions, suggesting that vincristine causes a hyperpolarization of the plasma membrane.

Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate.

An L1210 cell line (JT-1), which can grow in medium supplemented with 1 nM folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37 degrees C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23 +/- 0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [3H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37 degrees C remained as unmetabolized folic acid. Binding was also rapid at 0 degrees C but uptake at the plateau was only one-half the value obtained at 37 degrees C. Half-maximal saturation of the binding component (KD) occurred at a folate concentration of 0.065 nM at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (KD = 2.0 nM). 5-Methyltetrahydrofolate was also bound by this component (Ki = 13 nM at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (Ki = 45 nM) and methotrexate (Ki = 325 nM). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500 nM caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Methotrexate efflux in L1210 cells. Kinetic and specificity properties of the efflux system sensitive to bromosulfophthalein and its possible identity with a system which mediates the efflux of 3',5'-cyclic AMP.

Methotrexate exits L1210 mouse leukemia cells via multiple routes that include a unidirectional efflux component which is sensitive to bromosulfophthalein. This efflux component has been characterized in the present study after eliminating the contribution from the other efflux routes by treatment of the cells with an active ester of methotrexate and by reducing the assay pH to 6.2. The remaining efflux at pH 6.2 was greater than 90% sensitive to bromosulfophthalein. This route was also inhibited by probenecid, prostaglandin A1, diamide, 1-methyl-3-isobutylxanthine, various metabolic inhibitors, and by transfer of the cells to a buffer containing high concentrations of KCl. The inhibition by prostaglandin A1 was exceptionally potent and reached 50% at a concentration of 0.5 microM. An enhancement in efflux occurred upon the addition of glucose or by transfer of the cells to a non-saline buffer. When parameters relating to cellular energetics were measured, a reduction in ATP level was associated with the inhibition of efflux by probenecid, carbonylcyanide m-chlorophenylhydrazone, valinomycin, and antimycin A, whereas the increase in efflux by glucose was accompanied by an increase in intracellular ATP. Changes in ATP, however, were not associated with the inhibition by various other compounds or additions or with the enhancement in efflux by the non-anionic buffer. When the relative sensitivity of methotrexate efflux to bromosulfophthalein, 4,4'-diisothiocyanostilbene-2,2'-disulfonate, and lactic anhydride was compared with other anion transport systems, differences in specificity indicated that methotrexate was not exiting the cells via the bicarbonate/chloride exchange carrier, the lactate/H+ co-transport system, or a system which mediates the efflux of phthalate. However, a correlation was apparent between the sensitivity of methotrexate efflux to inhibition by prostaglandin A1, probenecid, and certain metabolic inhibitors and the ability of these same compounds to inhibit the unidirectional efflux of 3',5'-cyclic AMP in other cell lines, suggesting that methotrexate may share a common efflux route with cyclic nucleotides.

Transport of folate compounds by leukemic cells. Evidence for a single influx carrier for methotrexate, 5-methyltetrahydrofolate, and folate in CCRF-CEM human lymphoblasts.

Influx kinetics and inhibitor specificity have been compared for the transport of methotrexate, 5-methyltetrahydrofolate, and folate in CCRF-CEM human lymphoblastoid cells. Influx of each folate compound proceeded with approximately the same Vmax, fluctuated in the same fashion with the ionic composition of the medium, and was blocked by low concentrations of an N-hydroxysuccinimide ester of methotrexate in both an anion-deficient buffer and in a buffered saline medium containing physiological concentrations of glucose and bicarbonate. Moreover, methotrexate influx was inhibited by 5-methyltetrahydrofolate and folate, and the inhibition constants (Ki) of the latter compounds were equivalent to their Kt values for half-maximal influx. Folate influx was likewise inhibited by methotrexate. The Ki for methotrexate was equivalent to its Kt for influx, and o-phthalate and phosphate each inhibited folate and methotrexate with the same degree of effectiveness. Various reversible and irreversible inhibitors reduced the influx of each folate substrate by greater than 90%, and the progression of inhibition in each case was indicative of a single uptake component. Folate influx exhibited the same high sensitivity to inhibitors of methotrexate influx when measurements were performed at folate concentrations near the Kt for influx (10-50 microM) or at concentrations approximating physiological conditions (5-20 nM). These results indicate that CCRF-CEM cells possess a single shared transport system for the uptake of methotrexate, 5-methyltetrahydrofolate, and folate and that other high- or low-affinity uptake processes are not present in these cells.

Folate transport in Lactobacillus salivarius. Characterization of the transport mechanism and purification and properties of the binding component.

Lactobacillus salivarius cells contain an inducible transport system for folate. Influx via this system is time- and temperature-dependent, requires glucose and glutamine for optimum activity, and is half-maximal at folate concentrations in the nanomolar range. The folate internalized after 30 min at 30 degrees C is not released from the cells by excess extracellular folate and is recovered in cell extracts primarily in metabolized forms. A membrane-associated folate-binding protein is also present in cells that have been induced to transport folate. This binding protein constitutes 1% of total cellular protein, exhibits a high affinity for folate (KD = 0.40 nM), and requires divalent cations for optimum binding activity. Folate binds rapidly to this protein, while the exchange of bound substrate with folate added subsequently is relatively slow and dependent on the metabolic state of the cell. The transport rate per binding site is 0.05/min at 30 degrees C. A comparison of substrate specificity showed that folate binding and transport are both inhibited to the same extent by several different folate compounds, and a parallel irreversible inhibition of both processes is observed by prior treatment of the cells with a carbodiimide-activated derivative of folic acid. Binding protein labeled covalently with [3H]folate and solubilized with Triton X-100 was purified by a fractionation procedure involving absorption and elution from microgranular silica and molecular sieve chromatography. The isolated protein appeared homogeneous by gel electrophoresis and had an apparent molecular weight of 21,000. Monoclonal antibodies to the folate transport protein of Lactobacillus casei showed a high degree of cross-reactivity to the isolated binding protein from L. salivarius, indicating that these proteins share common epitopes. These results suggest that folate uptake by L. salivarius proceeds via an abundant membrane-associated binding protein which facilitates the movement of folate across the membrane as an electroneutral complex with cations. The substrate then slowly dissociates from internalized binding sites and is metabolized sequentially to coenzyme forms and then to membrane-impermeable folylpolyglutamates.

Characterization of the individual transport routes that mediate the influx and efflux of methotrexate in CCRF-CEM human lymphoblastic cells.

The transport routes utilized by CCRF-CEM human lymphoblastic cells for the influx and efflux of methotrexate have been analyzed. Evidence was obtained for a single influx route for methotrexate: (a) Influx at 2 microM [3H]methotrexate was inhibited completely by high concentrations of unlabeled methotrexate, o-phthalate, and bromosulfophthalein, and the inhibition profile with each anion was monophasic; and (b) Pretreatment of the cells with an N-hydroxysuccinimide ester of methotrexate also blocked influx, and this inhibition was complete over a range of substrate concentrations from 2 to 50 microM. Influx was also saturable and proceeded with a maximum rate (Vmax) of 4.3 pmol/min/mg protein (at 37 degrees C) and with a Kt of 0.8 microM in an anion-deficient buffer and 4.6 microM in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonate-buffered saline. The ratio of Vmax to the amount of carrier protein (0.3 pmol/mg protein) gave a turnover number for the transport system of 14.3/min. In contrast to influx, methotrexate efflux proceeded via three routes which could be separated by their sensitivity to specific inhibitors. The major portion of efflux occurred via the methotrexate influx carrier, the identity of which was established from its sensitivity to the N-hydroxysuccinimide ester of methotrexate and by its requirement for anions in the external medium. Methotrexate, adenosine monophosphate, and phosphate each stimulated efflux via this route and this stimulation was half-maximal at anion concentrations that approximated their Ki values for inhibition of methotrexate influx. A second efflux route was identified by its sensitivity to bromosulfophthalein. This route was relatively inactive and did not fluctuate significantly upon addition of various anions, glucose, or metabolic inhibitors. The third route was quantitated by its sensitivity to probenecid and its activity was increased in saline buffers and upon addition of glucose and was inhibited by oligomycin. Similar transport routes for methotrexate are present in L1210 mouse cells, although these two cell lines can be distinguished by the amount of transport protein and by the activity of the bromosulfophthalein-sensitive efflux route for methotrexate.