A comparative study on recurrent blooms of Alexandrium minutum in two Mediterranean coastal areas

Alexandrium minutum is a toxic dinoflagellate widespread along the Mediterranean coasts. This species is frequently detected year-round at low concentrations within the Mediterranean basin. However, it only proliferates recurrently in some localities. Two affected areas are the Catalan and Sicilian coasts. In order to identify the factors determining the A. minutum blooms in the Mediterranean Sea, we compare the bloom conditions in two harbours: Arenys de Mar (Catalan coast, Spain) and Syracuse (Sicily, Italy), during 2002–2003. Arenys de Mar harbour is a fishing and leisure harbour and receives an input of freshwater rich in nutrients. Likewise, the Syracuse harbour – located on the Ionian coast of Sicily – is subject to freshwater inputs. Some points of this site are used for productive activities such as shellfish farming. A. minutum from the two areas studied were morphologically and genetically identical. In both sites, recurrent blooms take place from winter to spring. Surface water temperatures and salinities during A. minutum bloom events were 12–14.5 °C and 32–38, and 16–24 °C and 32–37.7 for Arenys and Syracuse, respectively. During the blooms, the spatial distribution of A. minutum in the two harbours, the physicochemical characteristics and the phytoplankton community were studied. Similarities in composition of the phytoplankton community were evidenced, with a clear dominance of dinoflagellates over the other taxa. In Arenys, the second dominant species was Prorocentrum micans followed by Scrippsiella spp. and Dinophysis sacculus. The same species were found in Syracuse although P. triestinum, and alternatively Lingulodinium polyedrum, reached cell densities much higher than the other dinoflagellates giving marked water discolourations.     

Designing bryophyte surveys for an optimal coverage of diversity gradients

Knowledge on the distribution and abundance of species is plagued by significant taxonomic and geographic biases that influence the analyses on biodiversity patterns. Due to this, standard, easy-to-use methods are needed to design efficient field campaigns that minimize data deficiencies. We evaluate the applicability, usefulness and effectiveness of a survey design protocol based on the Environmental Diversity (ED) criterion under different scenarios, with examples of varying extent of environmental niche, range of spatial distribution and level of previous knowledge. We planned surveys for epiphytic bryophytes growing in three types of forests at NW Iberian Peninsula (dominated by Quercus ilex, Q. faginea and Q. pyrenaica). Knowledge on the distribution and abundance of epiphytic bryophytes in this region presents large gaps and strong geographic biases. Besides, the three forest types differ in their environmental requirements, spatial distribution and level of previous knowledge, providing three working scenarios to test the response of the protocol under different situations. The protocol was implemented as a set of sequential selection rules, starting by an ED-based criterion aiming at maximizing the coverage of climatic and geographic variability; further criteria include an iterative set of qualitative properties: maximizing forest area, conservation status and accessibility. The protocol performed efficiently at different ranges of spatial distribution levels of environmental variability, and degree of previous knowledge and generated an even distribution of sampling points that efficiently covered the diversity of epiphytic bryophytes. The results show that ED protocols are a proficient and time-saving approach to select sampling sites by objective criteria also for groups with high dispersal ability and fragmented landscapes.     

The C-terminal transmembrane domain of human phospholipid scramblase 1 is essential for the protein flip-flop activity and Ca2+-binding

Human phospholipid scramblase 1 (SCR) is a 318 amino acid protein that was originally described as catalyzing phospholipid transbilayer (flip-flop) motion in plasma membranes in a Ca²⁺-dependent, ATP-independent way. Further studies have suggested an intranuclear role for this protein in addition. A putative transmembrane domain located at the C terminus (aa 291–309) has been related to the flip-flop catalysis. In order to clarify the role of the C-terminal region of SCR, a mutant was produced (SCRΔ) in which the last 28 amino acid residues were lacking, including the α-helix. SCRΔ had lost the scramblase activity and its affinity for Ca²⁺ was decreased by one order of magnitude. Fluorescence and IR spectroscopic studies revealed that the C-terminal region of SCR was essential for the proper folding of the protein. Moreover, it was found that Ca²⁺ exerted an overall destabilizing effect on SCR, which might facilitate its binding to membranes.     

Multidrug Efflux Pump MdtBC of Escherichia coli Is Active Only as a B2C Heterotrimer

RND (resistance-nodulation-division) family transporters in Gram-negative bacteria frequently pump out a wide range of inhibitors and often contribute to multidrug resistance to antibiotics and biocides. An archetypal RND pump of Escherichia coli, AcrB, is known to exist as a homotrimer, and this construction is essential for drug pumping through the functionally rotating mechanism. MdtBC, however, appears different because two pump genes coexist within a single operon, and genetic deletion data suggest that both pumps must be expressed in order for the drug efflux to occur. We have expressed the corresponding genes, with one of them in a His-tagged form. Copurification of MdtB and MdtC under these conditions showed that they form a complex, with an average stoichiometry of 2:1. Unequivocal evidence that only the trimer containing two B protomers and one C protomer is active was obtained by expressing all possible combinations of B and C in covalently linked forms. Finally, conversion into alanine of the residues, known to form a proton translocation pathway in AcrB, inactivated transport only when made in MdtB, not when made in MdtC, a result suggesting that MdtC plays a different role not directly involved in drug binding and extrusion.Bacterial multidrug resistance is a major public health problem (10, 17). One widespread resistance mechanism involves the multidrug resistance (MDR) transporters. Among these, the resistance-nodulation-cell division (RND) family transporters, such as the AcrAB-TolC system in Escherichia coli, play a major role in drug resistance in Gram-negative bacteria because they allow the direct extrusion of drug molecules into extracellular space, and because they sometimes confer an increased level of tolerance to an astonishingly wide range of toxic compounds (18). In general, an RND-type exporter protein (such as AcrB), located in the inner membrane, forms a tripartite complex with a periplasmic adaptor protein, such as AcrA, and a homotrimeric outer membrane channel, such as TolC (18). The drug efflux process requires the presence of all three components. The crystallographic structures of AcrB (13, 14, 22, 24), AcrA (11, 27), and TolC (2, 8) are known, and models of the tripartite complex have been proposed (6, 27).AcrB is a homotrimeric transporter (14) located in the inner membrane and uses the proton gradient as the energy source (31). The homotrimeric structure is thought to be functionally important, or even essential, as each protomer appears to undergo a series of mandatory conformational alterations during the process of drug export, often called “functionally rotating mechanism,” as deduced from the structure of the asymmetric trimers of AcrB (13, 22, 24). This mechanism was also supported by the observation that, in a trimer in which protomers were covalently linked to each other, inactivation of one protomer alone abolishes the activity of the entire trimeric complex (29).Not all RND-type transporters, however, follow this homotrimeric organization. The mdtABC genes of E. coli encode an RND system that is unusual in that it contains two different RND pump genes, mdtB and mdtC, in addition to its own adaptor gene, mdtA. Previous genetic studies have demonstrated that the deletion of either of the two RND pump genes abolishes (1) the resistance to β-lactams, novobiocin, and bile salt derivatives, like deoxycholate, or narrows the range of pump substrates (15), a result suggesting that the functional unit is likely a heteromultimeric pump formed by MdtB/MdtC proteins. However, no direct data have so far been presented supporting the interaction between these proteins or the stoichiometry of the complex. Because the heterooligomeric composition of this pump was unexpected based on the accepted notion of how the homotrimeric pump functions by the functionally rotating mechanism, we examined here the nature of the MdtBC complex in detail.In this study, we first purified the oligomeric transporter by labeling either MdtB or MdtC with a His tag. We obtained a trimeric complex(es) containing both MdtB and MdtC in an approximately 2:1 ratio. However, we could not rule out the possibility that there were mixtures of trimers containing different ratios of the B and C proteins. We therefore utilized the recently introduced technology of creating covalently linked trimers (29), and we show here that the only active trimers are those containing two units of MdtB and one unit of MdtC.     

Characterization of the N-terminal domain of NrtC, the ATP-binding subunit of ABC-type nitrate transporter of the cyanobacterium Phormidium laminosum

The N-terminal domain of NrtC, the ATP-binding subunit of nitrate/nitrite ABC-transporter in the cyanobacterium Phormidium laminosum, has been expressed in Escherichia coli as a histidine-tagged fusion protein (His(6)NrtC1). Binding of ATP to the pure His(6)NrtC1 was characterized using the nucleotide analogue TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate]. Fluorescence assays showed that His(6)NrtC1 specifically binds Mg(2+) TNP-ATP with high affinity, binding being dependent on protein concentration. The presence of ATP prevents the covalent modification of His(6)NrtC1 by fluorescein 5'-isothiocyanate (FITC), suggesting that this probe reacts at the nucleotide-binding site of NrtC. The active form of the truncated NrtC is a dimer that shows high affinity for TNP-ATP (K(d)=0.76+/-0.1 microM). Evidence for the presence of two nucleotide-binding sites per dimer protein is given. Our results indicate that nucleotide binding is strongly dependent on the dimerization of NrtC and that the N-terminal domain of the protein contains the binding site for ATP. No ATPase activity catalyzed in vitro by the truncated subunit was detected.     

Visualization by High Resolution Immunoelectron Microscopy of the Transient Receptor Potential Vanilloid-1 at Inhibitory Synapses of the Mouse Dentate Gyrus

We have recently shown that the transient receptor potential vanilloid type 1 (TRPV1), a non-selective cation channel in the peripheral and central nervous system, is localized at postsynaptic sites of the excitatory perforant path synapses in the hippocampal dentate molecular layer (ML). In the present work, we have studied the distribution of TRPV1 at inhibitory synapses in the ML. With this aim, a preembedding immunogold method for high resolution electron microscopy was applied to mouse hippocampus. About 30% of the inhibitory synapses in the ML are TRPV1 immunopositive, which is mostly localized perisynaptically (∼60% of total immunoparticles) at postsynaptic dendritic membranes receiving symmetric synapses in the inner 1/3 of the layer. This TRPV1 pattern distribution is not observed in the ML of TRPV1 knock-out mice. These findings extend the knowledge of the subcellular localization of TRPV1 to inhibitory synapses of the dentate molecular layer where the channel, in addition to excitatory synapses, is present.     

The nitrate/nitrite ABC transporter of Phormidium laminosum: Phosphorylation state of NrtA is not involved in its substrate binding activity

Most cyanobacteria take up nitrate or nitrite through a multisubunit ABC transporter (ATP-binding cassette) located in the cytoplasmic membrane. Nitrate and nitrite transport activity is instantaneously blocked by the presence of ammonium in the medium. Previous biochemical studies reported the existence of phosphorylation/dephosphorylation events of the nitrate transporter (NRT) related to the presence of ammonium-sensitive kinase/phosphatase activities in plasma membranes of the cyanobacterium Synechococcus elongatus PCC 6301. In this work, we have analyzed the biochemical properties of the periplasmic nitrate/nitrite-binding subunit (NrtA) of NRT from the thermophilic nondiazotrophic cyanobacterium Phormidium laminosum. Our results show that cyanobacterial NrtA is phosphorylated in vivo. However, substrate binding activity in vitro is not affected by the phosphorylation state of the protein, ruling out the possibility that phosphorylation/dephosphorylation of NrtA is involved in the regulation of the nitrate/nitrite uptake by NRT transporter. Moreover, NrtA is present as multiple isoforms showing the same molecular mass but different isoelectric points ranging from pI 5 to 6. Mass spectrometric characterization of NrtA isoforms shows that the protein is phosphorylated at residue Tyr²⁰³, and contains several methionine sulphoxide residues which account for the observed isoforms. Both phosphorylated and non-phosphorylated forms of NrtA are active in vitro, showing comparable binding affinity for nitrate and nitrite. Both substrates behave as pure competitive inhibitors with a binding stoichiometry of one molecule of anion per NrtA monomer.