Sodium-dependent succinate uptake in purple bacterium Ectothiorhodospira shaposhnikovii

Succinate, malate and fumarate uptake in purple sulfur bacterium Ectothiorhodospira shaposhnikovii, strain 1 K MSU, obligatorily depends on the presence of Na⁺. Other monovalent cations such as K⁺, Li⁺, NH₄⁺ could not replace Na⁺. Experiments with energy-depleted cells have shown that succinate uptake against its concentration gradient can be energized by artificially imposed sodium gradients (ΔpNa).An artificial membrane potential (inside negative) inhibited ΔpNa-driven succinate uptake at pH 7.0 but stimulated it at pH 9.0.The results confirm the suggestion that succinate uptake in E. shaposhnikovii is carried out in symport with Na⁺.     

A novel N-linked flagellar glycan from Methanococcus maripaludis

The archaea Methanococcus maripaludis strain Mm900 produces flagella that are glycosylated with an N-linked tetrasaccharide. Mass spectrometric analysis of flagellar tryptic peptides identified a number of tryptic glycopeptides carrying a glycan of mass 1036.4 Da, and fragmentation of the glycan oxonium ion indicated that the glycan was a tetrasaccharide. The glycan was purified, following extensive pronase digestion of flagellar filaments, by size-exclusion and anion-exchange chromatography. NMR spectroscopy revealed that the glycan had the following structure:Sug-4-β-ManNAc3NAmA6Thr-4-β-GlcNAc3NAcA-3-β-GalNAc-Asnwhere Sug is a novel monosaccharide unit, (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-l-erythro-hexos-5-ulo-1,5-pyranose. This oligosaccharide has significant similarity to the oligosaccharide that was found previously in Methanococcus voltae.     

Methionine transport in the malaria parasite Plasmodium falciparum

The intraerythrocytic malaria parasite, Plasmodium falciparum, derives amino acids from the digestion of host cell haemoglobin. However, it also takes up amino acids from the extracellular medium. Isoleucine is absent from adult human haemoglobin and an exogenous source of isoleucine is essential for parasite growth. An extracellular source of methionine is also important for the normal growth of at least some parasite strains. In this study we have characterised the uptake of methionine by P. falciparum-infected human erythrocytes, and by parasites functionally isolated from their host cells by saponin-permeabilization of the erythrocyte membrane. Infected erythrocytes take up methionine much faster than uninfected erythrocytes, with the increase attributable to the flux of this amino acid via the New Permeability Pathways induced by the parasite in the erythrocyte membrane. Having entered the infected cell, methionine is taken up by the intracellular parasite via a saturable, temperature-dependent process that is independent of ATP, Na⁺ and H⁺. Substrate competition studies, and comparison of the transport of methionine with that of isoleucine and leucine, yielded results consistent with the hypothesis that the parasite has at its surface one or more transporters which mediate the flux into and out of the parasite of a broad range of neutral amino acids. These transporters function most efficiently when exchanging one neutral amino acid for another, thus providing a mechanism whereby the parasite is able to import important exogenous amino acids in exchange for surplus neutral amino acids liberated from the digestion of host cell haemoglobin.     

Structure and Function of the FeoB G-Domain from Methanococcus jannaschii

FeoB in bacteria and archaea is involved in the uptake of ferrous iron (Fe²⁺), an important cofactor in biological electron transfer and catalysis. Unlike any other known prokaryotic membrane protein, FeoB contains a GTP-binding domain at its N-terminus. We determined high-resolution X-ray structures of the FeoB G-domain from Methanococcus jannaschii with and without bound GDP or Mg²⁺-GppNHp. The G-domain forms the same dimer in all three structures, with the nucleotide-binding pockets at the dimer interface, as in the ATP-binding domain of ABC transporters. The G-domain follows the typical fold of nucleotide-binding proteins, with a β-strand inserted in switch I that becomes partially disordered upon GTP binding. Switch II does not contact the nucleotide directly and does not change its conformation in response to the bound nucleotide. Release of the nucleotide causes a rearrangement of loop L6, which we identified as the G5 region of FeoB. Together with the C-terminal helix, this loop may transmit the information about the nucleotide-bound state from the G-domain to the transmembrane region of FeoB.     

Structural characteristics of methanogenic cofactors in the non-methanogenic archaebacterium Archaeoglobus fulgidus

Archaeoglobus fulgidus is an extremely thermophilic, sulphate reducing archaebacterium thought to represent a biochemical missing-link between sulphur-metabolizing bacteria and methanogenic bacteria. Whereas the phylogenetic position of A.fulgidus is closer to the sulphur-metabolizing bacteria, there is a partial overlap in the biochemical machinery of A.fulgidus with both groups of bacteria. In particular, the presence of a number of aberrant cofactors up to now thought to be involved exclusively in the process of methanogenesis in methanogenic archaebacteria, i.e. coenzyme F420, methanofuran and methanopterin, has been indicated by previous studies. Here we present evidence for the structural identity of the methanopterin cofactor of A.fulgidus with the methanopterin isolated from Methanobacterium thermoautotrophicum and show that this non-methanogenic bacterium contains two as yet unknown analogues of coenzyme F420. The levels of the various cofactors were determined in cultures grown either on formate or lactate as the carbon source and sulphate or thiosulphate as the sulphur source.     

Crystal Structure of the Antitoxin-Toxin Protein Complex RelB-RelE from Methanococcus jannaschii

Here we present the crystal structure of the Methanococcus jannaschii RelE-RelB (RelBE) toxin-antitoxin (TA) protein complex determined by the MIRAS (multiple isomorphous replacement with anomalous signal) method. The genes encoding this TA system are located in the chromosome of this archaeon and involved in stress response. RelE acts as an endoribonuclease that cleaves mRNA on the ribosome, and we compare the RelBE complex to the known structures of other TA systems belonging to this group and to endoribonucleases. M. jannaschii RelBE forms a heterotetramer with the antitoxin in the centre of the complex, a configuration that differs vastly from the heterotetramer structure of the previously published RelBE from another archaeon, Pyrococcus horikoshii. The long N-terminal α-helix of the tightly bound M. jannaschii antitoxin RelB covers the presumed active site of the toxin RelE that is formed by a central β-sheet, a loop on one side and a C-terminal α-helix on the other side. The active site of the M. jannaschii toxin RelE harbours positive charges that are thought to neutralize the negative charges of the substrate mRNA, including Arg62 that was changed to Ser62 by the Escherichia coli expression system, thereby leading to inactive toxin RelE. Comparative studies suggest that Asp43 and His79 are also involved in the activity of the toxin.     

Glucose transport and metabolism in Gymnodinium breve

Florida's red tide organism, Gymnodinium breve, utilized exogenous glucose in the light for the synthesis of cellular components. Glucose was not taken up in the dark. Kinetic parameters for glucose uptake include a K_FD of 11 μM and a V_max of 1 × 10^?10 mol of glucose taken up/mg cellular protein/hr. Glucose uptake was competitively inhibited by phloridzin (K_i = 40 μM), mannose (K_i = 12O μM), and 2-deoxy-d-glucose (K_i = 190 μM) and non-competitively inhibited by galactose (K_i = 125 μM). Kinetics and inhibition of glucose uptake are consistent with a facilitated diffusion transport system.     

Asymmetry of glucose transport in the yeast, Kluyveromyces lactis

Uptake and efflux of 6-deoxy-d-[³H]glucose and of 2-deoxy-d-[¹⁴C]glucose by the yeast Kluyveromyces lactis was studied. The tritiated, nonphosphorylatable hexose analogue leaves the cell in the absence and presence of intracellular 2-deoxy-d-glucose 6-phosphate. In energy-rich cells containing pools of hexose 6-phosphate, 2-deoxy-d-glucose is trapped in the cells, for it neither effluxes into glucose-free medium nor exchanges with external, free sugar. In starved, poisoned cells containing negligible amounts of 2-deoxy-d-glucose 6-phosphate, 2-deoxy-d-glucose does leave the cells upon transfer to glucose-free medium. An involvement of analogue structure and availability of metabolites of energy-rich cells in hexose retention is suggested. An internal pool of 6-deoxy-d-glucose does not affect the rate of uptake of 6-deoxy-d-[³H]glucose, nor does internal 2-deoxy-d-[¹⁴C]glucose 6-phosphate influence that rate. Hence, transport of glucose by this yeast is probably not regulated by internal pools of glucose 6-phosphate.     

Shell formation and calcium transport in the barnacle Chthamalus fragilis

The mantle epithelium of the barnacle Chthamalus fragilis (Darwin) exhibits several ultrastructural features which may serve to regulate the calcification process. At the basis-mural plate and intermural plate junctions where rapid shell growth occurs, cells are characterized by long apical cytoplasmic projections and large intercellular spaces. These features may increase the functional surface area of the epithelium and enable more rapid deposition of calcium. The cells underlying the general shell surfaces contain numerous electron-dense inclusion bodies and show frequent cellular disintegration near the growing shell interface. Release of the granular contents of these inclusion bodies has been observed in both disintegrating and non-disintegrating cells. X-ray microanalysis revealed significantly higher calcium levels in the inclusion bodies than in the surrounding cytoplasm. This suggests a calcium transport role for these inclusion bodies. Cellular debris produced as a result of the disintegration of the mantle cells near the shell may play some role in the formation of the organic matrix of the shell. The presence of large numbers of mitochondria and well-developed apical microvilli in the cells of the inner mantle epithelium suggest that these cells serve to transport calcium into the mantle from the ambient sea water.     

Chelators in cupressaceae as iron transport agents for Salmonella typhimurium

Strains of Salmonella typhimurium which are unable to synthesize their own iron transport agents and require an erogenous chelator were used to examine extracts of the wood of species of Cupressaceae for the presence of iron chelators. Wood from 19 species of five genera were examined and all were found to contain substances that would function as iron transport agents for S. typhimurium. The biological activity of most of these species could be explained by the known presence and activity of the thujaplicins. Juniperus virginiana and J. occidentalis were found to contain a non-tropolone substance that functioned as chelators in S. typhimurium. The tropolone nootkatin from Chamaecyparis nootkatensis was ineffective as an iron transport agent.     

Acetate transport by locust rectum in vitro

Everted rectal sacs of Schistocerca gregaria absorb ¹⁴C-acetate from the lumen side at high rates against large electrical and often small concentration differences. Most of the ¹⁴C-activity in the absorbed fluid remains as acetate, but small amounts serve as substrate for aerobic respiration within this tissue. When acetate is substituted for SO₄^?2 or Cl^? in external salines, both short-circuit current (I_sc) and the open-circuit transepithelial potential (PD) increase by as much as 2- to 3-fold. The stimulatory effect of acetate on I_sc and PD exhibits saturation kinetics. The ‘steady-state’ influx of ¹⁴C-acetate from lumen (L) to haemocoel (H) side greatly exceeds efflux (haemocoel to lumen) across short-circuited recta. Over the whole range of acetate concentrations tested, the resulting net flux of acetate is sufficient to explain all of the increase in I_sc caused by this organic anion. Acetate was detected in moderate concentrations in body fluids of locusts. The possible significance of acetate transport in vivo is discussed.     

Ammonium (methylammonium) transport by Klebsiella pneumoniae

Klebsiella pneumoniae can accumulate methylammonium up to 80-fold by means of a transport system as indicated by the energy requirement, saturation kinetics and a narrow pH profile around pH 6.8. Methylammonium transport (apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 100 μ}\text{M}$, $\text{V = 40 μ}\text{mol/min}$ per g dry weight at 15°C) is competitively inhibited by ammonium (apparent $\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 7 μ}\text{M}$). The low $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ value and the finding that methylammonium cannot serve as a nitrogen source indicate that ammonium rather than methylammonium is the natural substrate. Uphill transport is driven by a component of the protonmotive force, probably the membrane potential. The transport system is under genetic control; it is partially repressed by amino acids and completely by ammonium. Analysis of mutants suggest that the synthesis of the ammonium transport system is subject to the same ‘nitrogen control’ as nitrogenase and glutamine synthetase.     

ATP is required for K+ active transport in the archaebacterium Haloferax volcanii

The Archaebacterium Haloferax volcanii concentrates K⁺ up to 3.6 M. This creates a very large K⁺ ion gradient of between 500- to 1,000-fold across the cell membrane. H. volcanii cells can be partially depleted of their internal K⁺ but the residual K⁺ concentration cannot be lowered below 1.5 M. In these conditions, the cells retain the ability to take up potassium from the medium and to restore a high internal K⁺ concentration (3 to 3.2 M) via an energy dependent, active transport mechanism with a K _m of between 1 to 2 mM. The driving force for K⁺ transport has been explored. Internal K⁺ concentration is not in equilibrium with m suggesting that K⁺ transport cannot be accounted for by a passive uniport process. A requirement for ATP has been found. Indeed, the depletion of the ATP pool by arsenate or the inhibition of ATP synthesis by N,N-dicyclohexylcarbodiimide inhibits by 100% K⁺ transport even though membrane potential m is maintained under these conditions. By contrast, the necessity of a m for K⁺ accumulation has not yet been clearly demonstrated. K⁺ transport in H. volcanii can be compared with K⁺ transport via the Trk system in Escherichia coli.Abbreviations CCCP Carbonylcyanide m-chlorophenyl-hydrazone - DCCD N,N-dicyclohexylcarbodiimide - MES 2-[N-morpholino] ethane sulfonic acid - MOPS 3-[N-morpholino] propane sulfonic acid - TRIS Tris (hydroxymethyl) aminomethane - TPP tetraphenyl phosphonium     

Sucrose transport by Streptococcus mutans. Evidence for multiple transport systems

The transport of sucrose by selected mutant and wild-type cells of Streptococcus mutans was studied using washed cocci harvested at appropriate phases of growth, incubated in the presence of fluoride and appropriately labelled substrates. The rapid sucrose uptake observed cannot be ascribed to possible extracellular formation of hexoses from sucrose and their subsequent transport, formation of intracellular glycogen-like polysaccharide, or binding of sucrose or extracellular glucans to the cocci. Rather, there are at least three discrete transport systems for sucrose, two of which are $\text{phospho}\text{enol}\text{pyruvate-dependent}$ phosphotransferases with relatively low apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values and the other a non-phosphotransferase (non-PTS) third transport system (termed TTS) with a relatively high apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. For strain 6715-13 mutant 33, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values are 6.25·10^?5 M, 2.4·10^?4 M, and 3.0·10^?3 M, respectively; for strain NCTC-10449, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values are 7.1·10^?5 M, 2.5·10^?4 M and 3.3·10^?3 M, respectively. The two lower $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ systems could not be demonstrated in mid-log phase glucose-adapted cocci, a condition known to repress sucrose-specific phosphotransferase activity, but under these conditions the highest $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ system persists. Also, a mutant devoid of sucrose-specific phosphotransferase activity fails to evidence the two high affinity (low apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$) systems, but still has the lowest affinity (highest $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$) system. There was essentially no uptake at 4°C indicating these processes are energy dependent. The third transport system, whose nature is unknown, appears to function under conditions of sucrose abundance and rapid growth which are known to repress $\text{phospho}\text{enol}\text{pyruvate-dependent}$ sucrose-specific phosphotransferase activity in S. mutans. These multiple transport systems seem well-adapted to S. mutans which is faced with fluctuating supplies of sucrose in its natural habitat on the surfaces of teeth.     

Amine transport at the plasmalemma of Riccia fluitans

Green thallus cells of the aquatic liverwort, Riccia fluitans, are rapidly depolarized in the presence of 1–20 μM NH₄Cl and 5–100 μM CH₃NH₃Cl, respectively. Simultaneously, the membrane conductance is increased from 0.41 to 1.2 S · m^?2. Uptake of [¹⁴C]methylamine is stimulated by increasing [K⁺]_o and inhibited by increasing [Na⁺]_o or [H⁺]_o, is highly voltage sensitive, and saturates at low amine concentrations.Double-reciprocal plots of (a) maximal membrane depolarization and (b) methylamine uptake vs. external amine concentration give apparent K_m values of 2 ± 1 μM ammonia and 25–50 μM methylamine; K_m values for changes in conductance and membrane current are greater and voltage dependent. Whereas the amine transport into the cell is strongly inhibited by CN^?, the amine efflux is stimulated.The current-voltage characteristics of the ammonia transport are represented by a sigmoid curve with an equilibrium potential of ?60 mV, and this is understood as a typical carrier curve with a saturation current of about 70 mA · m^?2. It is further concluded that the evidently carrier-mediated transport is competitive for the two amines tested, and that ammonia and methylamine are transported in the protonated form as NH₄⁺ and CH₃NH₃⁺ into the cytoplasm.     

Sodium-dependent transport of phosphate in LLC-PK1 cells

Transport of phosphate has been studied in subconfluent monolayers of LLC-PK₁ cells. It was found that this transport system shows similar characteristics to those observed in the kidney. Uptake of phosphate is mediated by a Na⁺-dependent, substrate-saturable process with an apparent K_m value for phosphate of 96 ± 15 μmol/1. Kinetic analysis of the effect of Na⁺ indicated that at (pH 7.4) two sodium ions are cotransported with one HPO₄^2? ion (Hill coefficient 1.5) with an apparent K_m value for sodium of 56 mmol/l. P_i uptake is inhibited by metabolic inhibitors (ouabain and FCCP). In the pH range of 6.6 of 7.4 P_i uptake rate does not change significantly, indicating that both the monovalent and the divalent form of phosphate are accepted by the transport system. It is suggested that phosphate is transported by LLC-PK_i cells together with sodium (2 Na⁺ :1 HPO₄^2?) in an electroneutral manner down a favourable sodium gradient.     

Primary structure and expression of a gene homologous to nifH (nitrogenase Fe protein) from the archaebacterium Methanococcus voltae

Summary In Methanococcus voltae, a 3.0 kbp HindIII fragment carrying homology to nifH was recently cloned. In Escherichia coli maxicells, the fragment directed the synthesis of a 30 K polypeptide encoded by the region homologous to nifH. Plasmids carrying the fragment did not complement Klebsiella pneumoniae nifH mutants and did not inhibit the nitrogen fixation of a Nif⁺ strain. The complete nucleotide sequence of the nifH homologous region was determined. It contained an open reading frame (ORFnifH) of 834 bp encoding 278 amino acid residues (mol. wt. 30,362). The ORFnifH was surrounded by regions of very high A+T content as observed with other mc. voltae genes. The region upstream from ORFnifH contained potential prokaryotic-like promoters and a potential ribosome binding site located 5 bp preceding the translation initiation codon. Using a translational fusion to lacZ of a DNA fragment carrying the putative promoter region and the 5 end of ORFnifH, it was shown in E. coli that (i) a promoter activity was effectively carried by the cloned fragment and (ii) this activity was not significantly modified by the presence of nifA or ntrC products provided by multicopy plasmids. Though the codon usage was characteristic of Mc. voltae, ORFnifH was very similar to eubacterial nifH genes, in particular the position of the cysteine residues was highly conserved. These data confirmed the high conservation of nifH sequences. S_AB values (binary matching coefficients) of 0.5 were found with eubacterial nifH genes at the nucleotide or amino acid level suggesting that the mc. voltae ORFnifH sequence was distantly related to eubacterial nifH sequences.     

l-Proline transport in Saccharomyces cerevisiae

Transport of l-proline into Saccharomyces cerevisiae K is mediated by two systems, one with a K_T of 31 μM and J_max of 40 nmol · s^?1 · (g dry wt.)^?1, the other with K_T > 2.5 mM and J_max of 150–165 nmol · s^?1 · (g dry wt.)^?1, The kinetic properties of the high-affinity system were studied in detail. It proved to be highly specific, the only potent competitive inhibitors being (i) l-proline and its analogs l-azetidine-2-carboxylic acid, sarcosine, d-proline and 3,4-dehydro-dl-proline, and (ii) l-alanine. The other amino acids tested behaved as noncompetitive inhibitors. The high-affinity system is active, has a sharp pH optimum at 5.8–5.9 and, in an Arrhenius plot, exhibits two inflection points at 15°C and 20–21°C. It is trans-inhibited by most amino acids (but probably only the natural substrates act in a trans-noncompetitive manner) and its activity depends to a considerable extent on growth conditions. In cells grown in a rich medium with yeast extract maximum activity is attained during the stationary phase, on a poor medium it is maximal during the early exponential phase. Some 50–60% of accumulated l-proline can leave cells in 90 min (and more if washing is done repeatedly), the efflux being insensitive to 0.5 mM 2,4-dinitrophenol and uranyl ions, to pH between 3 and 7.3, as well as to the presence of 10–100 mM unlabeled l-proline in the outside medium. Its rate and extent are increased by 1% d-glucose and by 10 μg nystatin per ml.