Metal stresses modify soluble proteomes and toxin profiles in two Mediterranean strains of the distributed dinoflagellate Alexandrium pacificum.

HABs involving Alexandrium pacificum have been reported in metal-contaminated ecosystems, suggesting that this distributed species adapts to and/or can tolerate the effects of metals. Modifications in soluble proteomes and PST contents were characterized in two Mediterranean A. pacificum strains exposed to mono- or polymetallic stresses (zinc, lead, copper, cadmium). These strains were isolated from two anthropized locations: Santa Giusta Lagoon (Italy, SG C10-3) and the Tarragona seaport (Spain, TAR C5-4F). In both strains, metals primarily downregulated key photosynthesis proteins. Metals also upregulated other proteins involved in photosynthesis (PCP in both strains), the oxidative stress response (HSP 60, proteasome and SOD in SG C10-3; HSP 70 in TAR C5-4F), energy metabolism (AdK in TAR C5-4F), neoglucogenesis/glycolysis (GAPDH and PEP synthase in SG C10-3) and protein modification (PP in TAR C5-4F). These proteins, possibly involved in adaptive proteomic responses, may explain the development of these A. pacificum strains in metal-contaminated ecosystems. The two strains showed different proteomic responses to metals, with SG C10-3 upregulating more proteins, particularly PCP. Among the PSTs, regardless of the metal and the strain studied, C2 and GTX4 predominated, followed by GTX5. Under the polymetallic cocktail, (i) total PSTs, C2 and GTX4 reached the highest levels in SG C10-3 only, and (ii) total PSTs, C2, GTX5 and neoSTX were higher in SG C10-3 than in TAR C5-4F, whereas in SG C10-3 under copper stress, total PSTs, GTX5, GTX1 and C1 were higher than in the controls, revealing variability in PST biosynthesis between the two strains. Total PSTs, C2, GTX4 and GTX1 showed significant positive correlations with PCP, indicating that PST production may be positively related to photosynthesis. Our results showed that the A. pacificum strains adapt their proteomic and physiological responses to metals, which may contribute to their ecological success in highly anthropized areas.

Nitrate supply and uptake in the Atlantic Arctic sea ice zone: seasonal cycle, mechanisms and drivers.

Nutrient supply to the surface ocean is a key factor regulating primary production in the Arctic Ocean under current conditions and with ongoing warming and sea ice losses. Here we present seasonal nitrate concentration and hydrographic data from two oceanographic moorings on the northern Barents shelf between autumn 2017 and summer 2018. The eastern mooring was sea ice-covered to varying degrees during autumn, winter and spring, and was characterized by more Arctic-like oceanographic conditions, while the western mooring was ice-free year-round and showed a greater influence of Atlantic water masses. The seasonal cycle in nitrate dynamics was similar under ice-influenced and ice-free conditions, with biological nitrate uptake beginning near-synchronously in early May, but important differences between the moorings were observed. Nitrate supply to the surface ocean preceding and during the period of rapid drawdown was greater at the ice-free more Atlantic-like western mooring, and nitrate drawdown occurred more slowly over a longer period of time. This suggests that with ongoing sea ice losses and Atlantification, the expected shift from more Arctic-like ice-influenced conditions to more Atlantic-like ice-free conditions is likely to increase nutrient availability and the duration of seasonal drawdown in this Arctic shelf region. The extent to which this increased nutrient availability and longer drawdown periods will lead to increases in total nitrate uptake, and support the projected increases in primary production, will depend on changes in upper ocean stratification and their effect on light availability to phytoplankton as changes in climate and the physical environment proceed. This article is part of the theme issue 'The changing Arctic Ocean: consequences for biological communities, biogeochemical processes and ecosystem functioning'.

The challenge of intracellular antibiotic accumulation, a function of fluoroquinolone influx versus bacterial efflux.

With the spreading of antibiotic resistance, the translocation of antibiotics through bacterial envelopes is crucial for their antibacterial activity. In Gram-negative bacteria, the interplay between membrane permeability and drug efflux pumps must be investigated as a whole. Here, we quantified the intracellular accumulation of a series of fluoroquinolones in population and in individual cells of Escherichia coli according to the expression of the AcrB efflux transporter. Computational results supported the accumulation levels measured experimentally and highlighted how fluoroquinolones side chains interact with specific residues of the distal pocket of the AcrB tight monomer during recognition and binding steps.

Mechanistic aspects of maltotriose-conjugate translocation to the Gram-negative bacteria cytoplasm.

Small molecule accumulation in Gram-negative bacteria is a key challenge to discover novel antibiotics, because of their two membranes and efflux pumps expelling toxic molecules. An approach to overcome this challenge is to hijack uptake pathways so that bacterial transporters shuttle the antibiotic to the cytoplasm. Here, we have characterized maltodextrin-fluorophore conjugates that can pass through both the outer and inner membranes mediated by components of the Escherichia coli maltose regulon. Single-channel electrophysiology recording demonstrated that the compounds permeate across the LamB channel leading to accumulation in the periplasm. We have also demonstrated that a maltotriose conjugate distributes into both the periplasm and cytoplasm. In the cytoplasm, the molecule activates the maltose regulon and triggers the expression of maltose binding protein in the periplasmic space indicating that the complete maltose entry pathway is induced. This maltotriose conjugate can (i) reach the periplasmic and cytoplasmic compartments to significant internal concentrations and (ii) auto-induce its own entry pathway via the activation of the maltose regulon, representing an interesting prototype to deliver molecules to the cytoplasm of Gram-negative bacteria.

Antibiotics and efflux: combined spectrofluorimetry and mass spectrometry to evaluate the involvement of concentration and efflux activity in antibiotic intracellular accumulation.

Background: In Gram-negative bacteria, passing through the double membrane barrier to reach the inhibitory concentration inside the bacterium is a pivotal step for antibiotic activity. Spectrofluorimetry has been developed to follow fluoroquinolone accumulation inside bacteria using intrinsic bacterial fluorescence as an internal standard. However, adaptation for non-fluorescent antibiotics is needed; quantitative methods based on MS offer the possibility of expanding the detection range obtained by spectrofluorimetry. Objectives: To validate, with spectrofluorimetry, the use of MS to measure antibiotic accumulation in cells and to determine the relationship between antibiotic concentrations and the amount of intrabacterial accumulation in different efflux backgrounds on the same batch of molecules. Methods: Spectrofluorimetry was performed in parallel with MS on the same samples to measure the ciprofloxacin and fleroxacin accumulation in cells expressing various efflux pump levels. A microplate protocol was set up to determine the antibiotic accumulation as a function of external antibiotic concentrations. Results: A correlation existed between the data obtained with spectrofluorimetry and MS, whatever the efflux pump or tested antibiotic. The results highlighted different dynamics of uptake between ciprofloxacin and fleroxacin as well as the relationship between the level of efflux activity and antibiotic accumulation. Conclusions: We have developed a microplate protocol and cross-validated two complementary methods: spectrofluorimetry, which contains a reliable internal standard; and MS, which allows detection of low antibiotic amounts. These assays allow study of the dose effect and the efflux impact on the intrabacterial accumulation of antibiotics.

Interplay Between Membrane Permeability and Enzymatic Barrier Leads to Antibiotic-Dependent Resistance in Klebsiella Pneumoniae.

The interplay between membrane permeability alterations and the enzymatic barrier contributes to Klebsiella pneumoniae multidrug resistance. We assessed the specific effect of the efflux levels of the main efflux pumps (AcrAB and OqxAB), alone and associated with the loss of the main porins (OmpK35 and OMPK36), on the activity of various antibiotics by constructing a set of K. pneumoniae isogenic strains, including strains with plasmid-mediated ß-lactamases (DHA-1, CTX-M-15, and OXA-48). The two pumps contributed to intrinsic chloramphenicol resistance and AcrAB to that of nalidixic acid and cefoxitin, whereas they had no impact on the activity of the other 11 antibiotics tested. We confirmed the expulsion of these three antibiotics by the two overproduced pumps and that of tigecycline by overproduced AcrAB, and showed that overproduced AcrAB also expelled ertapenem, piperacillin, ceftolozane, and ceftazidime. The sole loss of porins did not significantly affect the activity of the tested antibiotics, except ertapenem. The effect of efflux increases and porin loss on ß-lactam activity was the highest in plasmid-mediated ß-lactamase-producing strains. Thus, DHA-1-producing strains became non-susceptible (NS) to (i) ertapenem when there was an increase in efflux or porin loss, (ii) imipenem and ceftazidime+avibactam when the two mechanisms were associated, and (iii) temocillin when AcrAB was overproduced. The CTX-M-15-producing strains became NS to (i) ertapenem when there was no porin, (ii) ceftolozane+tazobactam when there was either overproduced OqxAB or porin loss, and (iii) temocillin when AcrAB was overproduced. OXA-48-producing strains known to be NS to temocillin were also NS to ceftolozane and they became NS to imipenem when the two pumps were overproduced or there was porin loss. Overall, this study shows that the balance between influx and efflux differentially modulates the activity of the tested antibiotics, an important point for evaluating the activity of future antibiotics or new combinations.

Spectrofluorimetric quantification of antibiotic drug concentration in bacterial cells for the characterization of translocation across bacterial membranes.

The efficacy of antibacterial molecules depends on their capacity to reach inhibitory concentrations in the vicinity of their target. This is particularly challenging for drugs directed against Gram-negative bacteria, which have a complex envelope comprising two membranes and efflux pumps. Precise determination of the bacterial drug content is an essential prerequisite for drug development. Here we describe three approaches that have been developed in our laboratories to quantify drugs accumulated in intact cells by spectrofluorimetry, microspectrofluorimetry, and kinetics microspectrofluorimetry (KMSF). These different procedures provide complementary results that highlight the contribution of membrane-associated mechanisms, including influx through the outer membrane (OM) and efflux, and the importance of the physicochemical properties of the transported drugs for the intracellular concentration of a given antibiotic in a given bacterial population. The three key stages of this protocol are preparation of the bacterial strains in the presence of the antibiotic; preparation of the whole-cell lysates (WCLs) and fluorescence readings; and data analysis, including normalization and quantitation of the intracellular antibiotic fluorescence relative to the internal standard and the antibiotic standard curve, respectively. Fluorimetry is limited to naturally fluorescent or labeled compounds, but in contrast to existing alternative methods such as mass spectrometry, it uniquely allows single-cell analysis. From culture growth to data analysis, the protocol described here takes 5 d.

Fluorescence enlightens RND pump activity and the intrabacterial concentration of antibiotics.

To understand antibiotic resistance in Gram-negative bacteria, a key point is to investigate antibiotic accumulation, which is defined by influx and efflux. Several methods exist to evaluate membrane permeability and efflux pump activity, but they present disadvantages and limitations. An optimized spectrofluorimetric method using intrinsic tryptophan fluorescence as an internal standard, as well as a complementary microfluorimetric assay following time-course accumulation in intact individual cells, have been developed. Comparing the latter population and single cell approaches can lead to an understanding of phenotypic heterogeneity within a population. The two methodologies lead to determination of parameters, concentration, accumulation rates and localization that contribute to emerging concepts (RTC2T, SICAR) with the aim of identifying and detailing antibiotic chemotypes involved in influx/efflux.

Fluoroquinolone structure and translocation flux across bacterial membrane.

Bacterial multidrug resistance is a worrying health issue. In Gram-negative antibacterial research, the challenge is to define the antibiotic permeation across the membranes. Passing through the membrane barrier to reach the inhibitory concentration inside the bacterium is a pivotal step for antibacterial molecules. A spectrofluorimetric methodology has been developed to detect fluoroquinolones in bacterial population and inside individual Gram-negative bacterial cells. In this work, we studied the antibiotic accumulation in cells expressing various levels of efflux pumps. The assays allow us to determine the intracellular concentration of the fluoroquinolones to study the relationships between the level of efflux activity and the antibiotic accumulation, and finally to evaluate the impact of fluoroquinolone structures in this process. This represents the first protocol to identify some structural parameters involved in antibiotic translocation and accumulation, and to illustrate the recently proposed "Structure Intracellular Concentration Activity Relationship" (SICAR) concept.

Modifications of the soluble proteome of a mediterranean strain of the invasive neurotoxic dinoflagellate Alexandrium catenella under metal stress conditions.

The soluble proteome of the mediterranean strain ACT03 of the invasive neurotoxic dinoflagellate Alexandrium catenella exposed to lead or zinc at 6, 12 or 18µM (total concentrations), or under control conditions, was characterized by two-dimensional gel electrophoresis (2-DE). Zinc reduced (P<0.05) the total number of protein spots (-41%, -52% and -60%, at 6, 12 or 18µM, respectively). Besides, most of the proteins constituting the soluble proteome were down-regulated in response to lead or zinc stresses. These proteins were involved mainly in photosynthesis (20-37% for lead; 36-50% for zinc) (ribulose-1,5-bisphosphate carboxylase/oxygenase: RUBISCO; ferredoxin-NADP+ reductase: FNR; peridinin-chlorophyll a-protein: PCP), and in the oxidative stress response (29-34% for lead; 17-36% for zinc) (superoxide dismutase: SOD; proteasome α/ß subunits). These negative effects could be partly compensated by the up-regulation of specific proteins such as ATP-synthase ß subunit (+16.3 fold after exposure to lead at 12µM). Indeed, an increase in the abundance of ATP-synthase could enrich the ATP pool and provide more energy available for the cells to survive under metal stress, and make the ATP-synthase transport of metal cations out of the cells more efficient. Finally, this study shows that exposure to lead or zinc have a harmful effect on the soluble proteome of A. catenella ACT03, but also suggests the existence of an adaptative proteomic response to metal stresses, which could contribute to maintaining the development of this dinoflagellate in trace metal-contaminated ecosystems.

Microspectrofluorimetry to dissect the permeation of ceftazidime in Gram-negative bacteria.

A main challenge in chemotherapy is to determine the in cellulo parameters modulating the drug concentration required for therapeutic action. It is absolutely urgent to understand membrane permeation and intracellular concentration of antibiotics in clinical isolates: passing the membrane barrier to reach the threshold concentration inside the bacterial periplasm or cytoplasm is the pivotal step of antibacterial activity. Ceftazidime (CAZ) is a key molecule of the combination therapy for treating resistant bacteria. We designed and synthesized different fluorescent CAZ derivatives (CAZ*, CAZ**) to dissect the early step of translocation-accumulation across bacterial membrane. Their activities were determined on E. coli strains and on selected clinical isolates overexpressing ß-lactamases. The accumulation of CAZ* and CAZ** were determined by microspectrofluorimetry and epifluorimetry. The derivatives were properly translocated to the periplasmic space when we permeabilize the outer membrane barrier. The periplasmic location of CAZ** was related to a significant antibacterial activity and with the outer membrane permeability. This study demonstrated the correlation between periplasmic accumulation and antibiotic activity. We also validated the method for approaching ß-lactam permeation relative to membrane permeability and paved the way for an original matrix for determining "Structure Intracellular Accumulation Activity Relationship" for the development of new therapeutic candidates.

New amphiphilic neamine conjugates bearing a metal binding motif active against MDR E. aerogenes Gram-negative bacteria.

Structure of bacterial envelope is one of the major factors contributing to Gram negative bacterial resistance. To develop new agents that target the bacterial membranes, we synthesized, by analogy with our previous peptide conjugates, new amphiphilic 3',4',6-trinaphthylmethylene neamines functionalized at position 5 through a short spacer by a chelating group, tris(2-pyridylmethyl)amine (TPA) and di-(picolyl)amine (DPA) and tetraazacyclotetradecane (Cyclam). ESI+ mass spectrometry analyses showed that neither Zn(II)(NeaDPA) nor Cu(II)(NeaCyclam) were stable in the Mueller Hinton (MH) medium used for antibacterial assays. In contrast Zn(NeaTPA) was stable in the MH medium. Interestingly, in MH, the free ligand NeaTPA was found bound to zinc, the zinc salt being the most abundant salt in this medium. Thus, the antibacterial activities of all compounds were evaluated as free ligands against E. coli strains, wild type AG100 and E. aerogenes EA289 (a clinical MDR strain that overexpresses AcrAB-TolC efflux pump), as well as AG100A an AcrAB- E. coli strain and EA298 a TolC- derivative. NeaCyclam and Zn(NeaTPA) were by far the most efficient compounds active against resistant isolate EA289 with MICs in the range 16-4 and 4 µM, respectively, while usual antibiotics such as ß-lactams and phenicols were inactive (MICs > 128) and ciprofloxacin was at 64 µM. Zn(NeaTPA) and NeaCyclam were shown to target and permeabilize the outer membrane of EA289 by promoting the cleavage of nitrocefin by periplasmic ß-lactamase. Moreover, all the neamine conjugates were able to block the efflux of 1,2'-dinaphthylamine in EA289, by acting on the efflux transporter located in the inner membrane. These membranotropic properties contribute to explain the activities of these neamine conjugates toward the MDR EA289 strain.

Evolution, three-dimensional model and localization of truncated hemoglobin PttTrHb of hybrid aspen.

Thus far, research on plant hemoglobins (Hbs) has mainly concentrated on symbiotic and non-symbiotic Hbs, and information on truncated Hbs (TrHbs) is scarce. The aim of this study was to examine the origin, structure and localization of the truncated Hb (PttTrHb) of hybrid aspen (Populus tremula L. × tremuloides Michx.), the model system of tree biology. Additionally, we studied the PttTrHb expression in relation to non-symbiotic class1 Hb gene (PttHb1) using RNAi-silenced hybrid aspen lines. Both the phylogenetic analysis and the three-dimensional (3D) model of PttTrHb supported the view that plant TrHbs evolved vertically from a bacterial TrHb. The 3D model suggested that PttTrHb adopts a 2-on-2 sandwich of α-helices and has a Bacillus subtilis -like ligand-binding pocket in which E11Gln and B10Tyr form hydrogen bonds to a ligand. However, due to differences in tunnel cavity and gate residue (E7Ala), it might not show similar ligand-binding kinetics as in Bs-HbO (E7Thr). The immunolocalization showed that PttTrHb protein was present in roots, stems as well as leaves of in vitro -grown hybrid aspens. In mature organs, PttTrHb was predominantly found in the vascular bundles and specifically at the site of lateral root formation, overlapping consistently with areas of nitric oxide (NO) production in plants. Furthermore, the NO donor sodium nitroprusside treatment increased the amount of PttTrHb in stems. The observed PttTrHb localization suggests that PttTrHb plays a role in the NO metabolism.

Exploring chloroplastic changes related to chilling and freezing tolerance during cold acclimation of pea (Pisum sativum L.).

Pea (Pisum sativum L.) productivity is linked to its ability to cope with abiotic stresses such as low temperatures during fall and winter. In this study, we investigate the chloroplast-related changes occurring during pea cold acclimation, in order to further lead to genetic improvement of its field performance. Champagne and Térèse, two pea lines with different acclimation capabilities, were studied by physiological measurements, sub-cellular fractionation followed by relative protein quantification and two-dimensional DIGE. The chilling tolerance might be related to an increase in protein related to soluble sugar synthesis, antioxidant potential, regulation of mRNA transcription and translation through the chloroplast. Freezing tolerance, only observed in Champagne, seems to rely on a higher inherent photosynthetic potential at the beginning of the cold exposure, combined with an early ability to start metabolic processes aimed at maintaining the photosynthetic capacity, optimizing the stoichiometry of the photosystems and inducing dynamic changes in carbohydrate and protein synthesis and/or turnover.

Wheat CBF gene family: identification of polymorphisms in the CBF coding sequence.

Expression of cold-regulated genes needed for protection against freezing stress is mediated, in part, by the CBF transcription factor family. Previous studies with temperate cereals suggested that the CBF gene family in wheat was large, and that CBF genes were at the base of an important low temperature tolerance trait. Therefore, the goal of our study was to identify the CBF repertoire in the freezing-tolerant hexaploid wheat cultivar Norstar, and then to examine if the coding region of CBF genes in two spring cultivars contain polymorphisms that could affect the protein sequence and structure. Our analyses reveal that hexaploid wheat contains a complex CBF family consisting of at least 65 CBF genes of which 60 are known to be expressed in the cultivar Norstar. They represent 27 paralogous genes with 1-3 homeologous copies for the A, B, and D genomes. The cultivar Norstar contains two pseudogenes and at least 24 additional proteins having sequences and (or) structures that deviate from the consensus in the conserved AP2 DNA-binding and (or) C-terminal activation-domains. This suggests that in cultivars such as Norstar, low temperature tolerance may be increased through breeding of additional optimal alleles. The examination of the CBF repertoire present in the two spring cultivars, Chinese Spring and Manitou, reveals that they have additional polymorphisms affecting conserved positions in these domains. Understanding the effects of these polymorphisms will provide additional information for the selection of optimum CBF alleles in Triticeae breeding programs.

Protein expression from zooplankton communities in a metal contaminated NW mediterranean coastal ecosystem.

Bidimensional and monodimensional polyacrylamide gel electrophoresis were used to study protein expression from zooplankton collected in thirteen stations of Toulon Bay (NW Mediterranean). In this ecosystem, Little Bay showed higher trace metal concentrations (13.5-23.8 nM for Cu, 0.73-1.24 nM for Pb, 27.8-58.7 nM for Zn) than Large Bay (Cu 2.2-15.6 nM; Pb 0.19-0.78 nM; Zn 9.0-38.8 nM). Trace metals positively correlated (p < 0.05) with expression of four zooplankton proteins (MW in kDa/pI: 25.0/5.6; 48.8/4.1; 38.2/4.4; 38.3/5.8) and with biomass of Oithona nana, predominant copepod in Little Bay. Sequencing by LC-MS/MS putatively provided zooplankton identity of these proteins: they were cytoskeleton actin, except one protein that was the chaperone calreticulin. We suggest that actin and calreticulin could be regarded as zooplankton markers of metal stress and be involved in a possible tolerance of O. nana to contamination, contributing to its development in a marine perturbed ecosystem.

A proteomic approach to decipher chilling response from cold acclimation in pea (Pisum sativum L.).

Two pea lines (Pisum sativum L.) with contrasted behaviours towards chilling and subsequent frost were studied by a proteomic approach to better understand cold acclimation. Following a chilling period, the Champagne line becomes tolerant to frost whereas Terese remains sensitive. Variance analysis allowed to select 260 statistically variable spots with 68 identified proteins (35 in leaves, 18 in stems, and 15 in roots). These proteins were shared out in proteins related to chilling response or cold acclimation. The better adaptation of Champagne to chilling might be related to a higher content in proteins involved in photosynthesis and in defence mechanisms. Moreover Champagne might prevent freezing damage particularly thanks to a higher constitutive expression of housekeeping proteins related to Terese. After three days of subsequent frost, proteomes of previously chilled plants also showed significant differences compared to unchilled plants. Out of 112 statistically variable spots (44 in leaves, 38 in stems, and 30 in roots), 32 proteins were identified. These proteins were related to frost response or frost resistance. It seems that Champagne could resist to frost with the reorientation of the energy metabolism.

Association of sugar content QTL and PQL with physiological traits relevant to frost damage resistance in pea under field and controlled conditions.

To increase yield in pea (Pisum sativum L.), autumn sowing would be preferable. Hence, frost tolerance of pea became a major trait of interest for breeders. In order to better understand the cold acclimation in pea, Champagne a frost tolerant line and Terese, a frost sensitive line, and their recombinant inbred lines (RIL) were studied. RIL frost tolerance was evaluated by a frost damage scale under field as well as controlled conditions. A quantitative trait loci (QTL) approach was used to identify chromosomal regions linked to frost tolerance. The detected QTL explained from 6.5 to 46.5% of the phenotypic variance. Amongst them, those located on linkage groups 5 and 6 were consistent with over all experiments, in field as well as in controlled environments. In order to improve the understanding of the frost tolerance mechanisms, several cold acclimation key characters such as concentration of sugars, electrolyte leakage, osmotic pressure, and activity of RuBisCO were assessed. Some of these physiological QTL colocalised with QTL for frost damage, in particular two raffinose QTL on LG5 and LG6 and one RuBisCO activity QTL on LG6, explaining 8.8 to 27.0% of the phenotypic variance. In addition, protein quantitative loci were mapped; some of them colocalised with frost damage and physiological QTL on LG5 and LG6, explaining 16.0-43.6% of the phenotypic variance. Raffinose metabolism and RuBisCO activity and its effect on photosynthesis might play a major role in cold acclimation of pea.