Effect of cetrimonium carrier micelles on bacterial membranes and extracellular DNA, an in silico study.

Microorganisms do not live as dispersed single cells but rather they form aggregates with extracellular polymeric substances at interfaces. Biofilms are considered efficient life forms because they shield bacteria from biocides and collect dilute nutrients. This is a big concern in industry since the microorganisms can colonize a wide range of surfaces, accelerating material deterioration, colonizing medical devices, contaminating ultrapure drinking water, increasing energy costs and creating focus of infection. Conventional biocides that target a specific component of the bacteria are not effective in the presence of biofilms. Efficient biofilm inhibitors are based on a multitarget approach interacting with the bacteria and the biofilm matrix. Their rationale design requires a thorough understanding of inhibitory mechanisms that are still largely lacking today. Herein we uncover via molecular modelling the inhibition mechanism of cetrimonium 4-OH cinnamate (CTA-4OHcinn). Simulations show that CTA-4OH micelles can disrupt symmetric and asymmetric bilayers, representative of inner and outer bacterial membranes, following three stages: adsorption, assimilation, and defect formation. The main driving force for micellar attack is electrostatic interactions. In addition to disrupting the bilayers, the micelles work as carriers facilitating the trapping of 4OH cinnamate anions within the bilayer upper leaflet and overcoming electrostatic repulsion. The micelles also interact with extracellular DNA (e-DNA), which is one of the main components of biofilms. It is observed that CTA-4OHcinn forms spherical micelles on the DNA backbone; which hinders their ability to pack. This is demonstrated by modelling the DNA along the hbb histone-like protein, showing that in the presence of CTA-4OHcinn, DNA does not pack properly around hbb. The abilities of CTA-4OHcinn to cause cell death through membrane disruption and to disperse a mature, multi-species biofilm are also confirmed experimentally.

EMT Molecular Signatures of Pancreatic Neuroendocrine Neoplasms.

Neuroendocrine neoplasms (NENs) are relatively rare neoplasms occurring predominantly in the gastrointestinal tract and pancreas. Their heterogeneity poses challenges for diagnosis and treatment. There is a paucity of markers for characterisation of NEN tumours. For routine diagnosis, immunohistochemistry of the NEN-specific markers CgA and synaptophysin and the proliferation marker Ki-67 are used. These parameters, however, are qualitative and lack the capacity to fully define the tumour phenotype. Molecules of epithelial-mesenchymal transition (EMT) are potential candidates for improved tumour characterisation. Using qRT-PCR, we measured mRNA levels of 27 tumour markers, including 25 EMT-associated markers, in tumour tissue and matched non-tumour tissues for 13 patients with pancreatic NENs. Tissue from patients with three different grades of tumour had distinctly different mRNA profiles. Of the 25 EMT-associated markers analysed, 17 were higher in G3 tissue relative to matched non-tumour tissue, including CD14, CD24, CD31, CD44, CD45, CD56, CK6, CK7, CK13, CK20, NSE, CDX2, CgA, DAXX, PCNA, laminin and Ki-67. The differences in levels of seven EMT-associated markers, Ki-67, DAXX, CD24, CD44, vimentin, laminin and PDX1 plus CgA and NSE (neuroendocrine markers) enabled a distinct molecular signature for each tumour grade to be generated. EMT molecules differentially expressed in three tumour grades have potential for use in tumour stratification and prognostication and as therapeutic targets for treatment of neuroendocrine cancers, following validation with additional samples.

Review of the structures and functions of algal photoreceptors to optimize bioproduct production with novel bioreactor designs for strain improvement.

Microalgae are important renewable feedstock to produce biodiesel and high-value chemicals. Different wavelengths of light influence the growth and metabolic activities of algae. Recent research has identified the light-sensing proteins called photoreceptors that respond to blue or red light. Structural elucidations of algal photoreceptors have gained momentum over recent years. These include channelrhodopsins, PHOT proteins, animal-like cryptochromes, and blue-light sensors utilizing flavin-adenine dinucleotide proteins. Pulsing light has also been investigated as a means to optimize energy inputs into bioreactors. This study summarizes the current structural and functional basis of photoreceptor modulation to optimize the growth, production of carotenoids and other high-value metabolites from microalgae. The review also encompasses novel photobioreactor designs that implement different light regimes including light wavelengths and time to optimize algal growth and desired metabolite profiles for high-value products.

Genome Sequence of Lelliottia sp. Strain WAP21, Isolated from Soil in Canola Fields in Victoria, Australia.

Here, we describe the genome of Lelliottia sp. strain WAP21, which was isolated from the soil of canola fields in Australia. The genome has a size of 4.9 Mbp and 4,583 predicted genes, with some potential pathways for metabolism of various carbon sources and metal acquisition.

Modelling cetrimonium micelles as 4-OH cinnamate carriers targeting a hydrated iron oxide surface.

HYPOTHESIS: Molecular interactions between 4-OH-cinnamate and cetrimonium in solution result in improved adsorption of the cinnamate on mild steel, developing a protective mechanism against the diffusion of corrosive chloride to the oxide surface. Fundamental understanding of this mechanism should allow new design routes for the development of eco-friendly corrosion inhibitors. EXPERIMENTS: Via classic molecular dynamics, simulations were carried out for cetrimonium and 4-OH-cinnamate in aqueous solutions at different ionic strengths and the results were validated with experimental SAXS data. Self-aggregation of cetrimonium 4-OH-cinnamate on a hydrated hematite surface was then simulated and results were compared with cryo-TEM imaging for the same compound. Finally, the effect of the adsorbed aggregates on chloride diffusion to the oxide surface was modelled. FINDINGS: Simulations showed the encapsulation of 4-OH-cinnamate into cetrimonium micelles, consistent with experiments. The newly formed micelles adsorb onto a hydrated iron oxide surface by forming hydrogen bonds between their carboxylate outer-shell groups and the surface hydroxyls. As the adsorbate concentrations increase, there is a morphological transition from spherical to wormlike adsorbed aggregates. The wormlike structure can block chloride ions, demonstrating a synergistic inhibitory mechanism between both cetrimonium and 4-OH-cinnamate. Encapsulation and delivery of active compounds to certain targets, such as carcinogenic tumors, have been well studied in biochemistry research, we demonstrate that the same mechanism can be applied to the design of efficient corrosion inhibitors, optimizing their delivery to the metal surface.

mRNA profiling of a well-differentiated G1 pancreatic NET correlates with immunohistochemistry profile: a case report.

BACKGROUND: Neuroendocrine neoplasms (NENs) are a complex group of tumours that occur in many organs. Routinely used IHC markers for NEN diagnosis include CgA, synaptophysin, Ki67 and CD56. These have limitations including lack of correlation to clinical outcomes and their presence in non-tumour tissue. Identification of additional markers and more quantitative analyses of tumour tissue has the potential to contribute to improved clinical outcomes. We used qRT-PCR to profile the expression levels of a panel of markers in tumour and matched non-tumour tissue from a patient with a G1 pancreatic neuroendocrine tumour. Differences in mRNA levels between tumour and non-tumour tissue were compared with IHC analyses of the same sample. CASE PRESENTATION: An elderly man presented with lower abdominal pain for 6 months. Histological analysis identified a low grade, well differentiated pancreatic endocrine neoplasm. Twenty-seven tumour markers for neuroendocrine status, proliferation, stem cell phenotype, angiogenesis, epithelial to mesenchymal transition, cell adhesion, differentiation and tumour suppression were selected from previous studies and mRNA levels of these markers were measured in tumour and adjacent non-tumour tissue sample using qRT-PCR. IHC was carried out on the same tissue to detect the corresponding marker proteins. Of the markers analysed, seven showed higher mRNA levels in tumour relative to non-tumour tissue while thirteen had lower expression in tumour relative to non-tumour tissue. Substantial differences in mRNA levels were a gain of CgA, CD56, ß-catenin, CK20, PDX1 and p53 and loss of Ki67, PCAD, CK7, CD31, MENA, ECAD, EPCAM, CDX2 and CK6. Comparison of qRT-PCR data with IHC showed correlation between fifteen markers. CONCLUSION: Our study is unique as it included matched controls that provided a comparative assessment for tumour tissue analysis, whereas many previous studies report tumour data only. Additionally, we utilised qRT-PCR, a relatively quantitative diagnostic tool for differential marker profiling, having the advantage of being reproducible, fast, cheap and accurate. qRT-PCR has the potential to improve the defining of tumour phenotypes and, in combination with IHC may have clinical utility towards improving tumour stratification or distinguishing tumour grades. The results need to be validated with different grades of NENs and related to clinical outcomes.

Chromium tolerance and accumulation in Aspergillus flavus isolated from tannery effluent.

Cr(VI) tolerance in Aspergillus flavus, strain SFL, isolated from tannery effluent was measured and compared with a reference strain of A. flavus, A1120. On solid medium, SFL had a high level of Cr(VI) tolerance (1,600 mg/L), which was 16 times that of A1120 and greater than most previously analyzed fungal strains. When in 100 mg/L of Cr(VI), SFL completely depleted Cr(VI) within 72 h while A1120 depleted 85% of Cr(VI). SFL was more effective in reducing extracellular Cr(VI) than A1120. While A1120 showed greater biosorption of Cr(VI) than SFL, intracellular accumulation was approximately 50% greater in SFL and was more energy-dependent than A1120. Cr(VI) modified the external surface of the hyphae. Cr speciation detected the presence of only Cr(III), corresponding to Cr(OH)3 , which precipitated on the hyphal surface. Cr(VI) bound to the functional groups carboxyl, amine, and hydroxyl in both SFL and A1120. Transmission electron microscopy energy-dispersive X-ray detected Cr on the fungal wall and within membrane-bound organelles of the cytoplasm. In conclusion, the greater tolerance of SFL to Cr(VI) relative to A1120 is due to more effective energy-dependant uptake of Cr(VI) into the cell and increased capacity of SFL to store Cr in intracellular vacuoles compared with A1120.

Transient epigenomic changes during pregnancy and early postpartum in women with and without type 2 diabetes.

AIM: To investigate epigenomic changes in pregnancy and early postpartum in women with and without type 2 diabetes. METHODS: Dimethylation of histones H3K4, H3K9, H3K27, H3K36 and H3K79 was measured in white blood cells of women at 30 weeks pregnancy, at 8-10 and 20 weeks postpartum and in never-pregnant women. RESULTS: Dimethylation levels of all five histones were different between women in pregnancy and early postpartum compared with never-pregnant women and were different between women with and without type 2 diabetes. CONCLUSION: Histone methylation changes are transient in pregnancy and early postpartum and may represent normal physiological responses to hormones. Different epigenomic profiles in women with type 2 diabetes mellitus may correlate with hormonal responses, leading to high risk pregnancy outcomes.

Critical effects of polar fluorescent probes on the interaction of DHA with POPC supported lipid bilayers.

The understanding of lipid bilayer structure and function has been advanced by the application of molecular fluorophores. However, the effects of these probe molecules on the physicochemical properties of membranes being studied are poorly understood. A quartz crystal microbalance with dissipation monitoring instrument was used in this work to investigate the impact of two commonly used fluorescent probes, 1­palmitoyl­2­{12­[(7­nitro­2­1,3­benzoxadiazol­4­yl)amino]dodecanoyl}­sn­glycero­3­phosphocholine (NBD-PC) and 1,2­dipalmitoyl­sn­glycero­3­phosphoethanolamine­n­(lissamine rhodamine­B­sulfonyl) (Lis-Rhod PE), on the formation and physicochemical properties of a 1­palmitoyl­2­oleoyl­sn­glycero­3­phosphocholine supported lipid bilayer (POPC-SLB). The interaction of the POPC-SLB and fluorophore-modified POPC-SLB with docosahexaenoic acid, DHA, was evaluated. The incorporation of DHA into the POPC-SLB was observed to significantly decrease in the presence of the Lis-Rhod PE probe compared with the POPC-SLB. In addition, it was observed that the small concentration of DHA incorporated into the POPC:NBD-PC SLB can produce rearrangement processes followed by the lost not only of DHA but also of POPC or NBD-PC molecules or both during the washing step. This work has significant implications for the interpretation of data employing fluorescent reporter molecules within SLBs.

Identifying Epithelial Endocytotic Mechanisms of the Peanut Allergens Ara h 1 and Ara h 2.

BACKGROUND: Peanuts are still one of the highest contributors to anaphylactic deaths after ingestion of a food allergen. At the molecular level, interactions between peanut allergens and the intestinal epithelium are largely unexplored. Previous findings by our research group demonstrated that the major peanut allergens, i.e., Ara h 1, Ara h 2, Ara h 3, and Ara h 6, were able to cross the Caco-2 human cell culture model of the intestinal epithelium. This research broadened our investigation to identify the mechanisms by which the Caco-2 monolayers uptake peanut allergens, specifically by endocytosis. Here, we aim to increase our understanding of allergen-epithelial interactions and, more broadly, the pathway from allergen to allergy. METHODS: The human Caco-2 cell culture model was exposed to peanut extract and a combination of confocal microscopy and inhibition studies were used to identify the endocytotic mechanisms of peanut allergens in intestinal epithelia. RESULTS: Our findings demonstrate that the peanut allergens Ara h 1 and Ara h 2 are transported through intestinal epithelia initially via early endosomes using multiple endocytotic mechanisms. From there, they are then transported to late endosomes and ultimately to lysosomes. CONCLUSIONS: These novel findings provide insight into the allergen-epithelial interactions of peanut allergens with the intestinal epithelium. Consequently, this opens the possibility of the use of these endocytotic pathways as targets for inhibitors in therapeutic development and preventative measures for peanut allergy in the future.

Peroxide reduction by a metal-dependent catalase in Nostoc punctiforme (cyanobacteria).

This study investigated the role of a novel metal-dependent catalase (Npun_R4582) that reduces hydrogen peroxide in the cyanobacterium Nostoc punctiforme. Quantitative real-time PCR showed that npun_R4582 relative mRNA levels were upregulated by over 16-fold in cells treated with either 2 µM added Co, 0.5 µM added Cu, 500 µM Mn, 1 µM Ni, or 18 µM Zn. For cells treated with 60 µM H2O2, no significant alteration in Npun_R4582 relative mRNA levels was detected, while in cells treated with Co, Cu, Mn, Ni, or Zn and 60 µM peroxide, relative mRNA levels were generally above control or peroxide only treated cells. Disruption or overexpression of npun_R4582 altered sensitivity to cells exposed to 60 µM H2O2 and metals for treatments beyond the highest viable concentrations, or in a mixed metal solution for Npun_R4582- cells. Moreover, overexpression of npun_R4582 increased cellular peroxidase activity in comparison with wild-type and Npun_R4582- cells, and reduced peroxide levels by over 50%. The addition of cobalt, manganese, nickel, and zinc increased the capacity of Npun_R4582 to reduce the rate or total levels of peroxide produced by cells growing under photooxidative conditions. The work presented confirms the function of NpunR4582 as a catalase and provides insights as to how cells reduce potentially lethal peroxide levels produced by photosynthesis. The findings also show how trace elements play crucial roles as enzymatic cofactors and how the role of Npun_R4582 in hydrogen peroxide breakdown is dependent on the type of metal and the level available to cells.

Microbial nanowires: an electrifying tale.

Electromicrobiology has gained momentum in the last 10 years with advances in microbial fuel cells and the discovery of microbial nanowires (MNWs). The list of MNW-producing micro-organisms is growing and providing intriguing insights into the presence of such micro-organisms in diverse environments and the potential roles MNWs can perform. This review discusses the MNWs produced by different micro-organisms, including their structure, composition and mechanism of electron transfer through MNWs. Two hypotheses, metallic-like conductivity and an electron hopping model, have been proposed for electron transfer and we present a current understanding of both these hypotheses. MNWs not only are poised to change the way we see micro-organisms but also may impact the fields of bioenergy, biogeochemistry and bioremediation; hence, their potential applications in these fields are highlighted here.

Real-Time Quartz Crystal Microbalance Monitoring of Free Docosahexaenoic Acid Interactions with Supported Lipid Bilayers.

Docosahexaenoic acid (DHA) is the most abundant polyunsaturated omega-3 fatty acid found in mammalian neuronal cell membranes. Although DHA is known to be important for neuronal cell survival, little is know about how DHA interacts with phospholipid bilayers. This study presents a detailed quartz crystal microbalance with dissipation monitoring (QCM-D) analysis of free DHA interactions with individual and mixed phospholipid supported lipid bilayers (SLB). DHA incorporation and subsequent changes to the SLBs viscoelastic properties were observed to be concentration-dependent, influenced by the phospholipid species, the headgroup charge, and the presence or absence of calcium ions. It was observed that 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) SLBs incorporated the greatest amount of DHA concentration, whereas the presence of phospholipids, phosphatidylserine (PS), and phosphatidylinositol (PI) in a POPC SLB significantly reduced DHA incorporation and changed the SLBs physicochemical properties. These observations are hypothesized to be due to a substitution event occurring between DHA and phospholipid species. PS domain formation in POPC/PS 8:2 SLBs was observed in the presence of calcium ions, which favored DHA incorporation to a similar level as for a POPC only SLB. The changes in SLB thickness observed with different DHA concentrations are also presented. This work contributes to an understanding of the physical changes induced in a lipid bilayer as a consequence of its exposure to different DHA concentrations (from 50 to 200 µM). The capacity of DHA to influence the physical properties of SLBs indicates the potential for dietary DHA supplementation to cause changes in cellular membranes in vivo, with subsequent physiological consequences for cell function.

Probing Synechocystis-Arsenic Interactions through Extracellular Nanowires.

Microbial nanowires (MNWs) can play an important role in the transformation and mobility of toxic metals/metalloids in environment. The potential role of MNWs in cell-arsenic (As) interactions has not been reported in microorganisms and thus we explored this interaction using Synechocystis PCC 6803 as a model system. The effect of half maximal inhibitory concentration (IC50) [~300 mM As (V) and ~4 mM As (III)] and non-inhibitory [4X lower than IC50, i.e., 75 mM As (V) and 1 mM As (III)] of As was studied on Synechocystis cells in relation to its effect on Chlorophyll (Chl) a, type IV pili (TFP)-As interaction and intracellular/extracellular presence of As. In silico analysis showed that subunit PilA1 of electrically conductive TFP, i.e., microbial nanowires of Synechocystis have putative binding sites for As. In agreement with in silico analysis, transmission electron microscopy analysis showed that As was deposited on Synechocystis nanowires at all tested concentrations. The potential of Synechocystis nanowires to immobilize As can be further enhanced and evaluated on a large scale and thus can be applied for bioremediation studies.

Ceruloplasmin is regulated by copper and lactational hormones in PMC42-LA mammary epithelial cell culture models.

Ceruloplasmin (Cp) is a multicopper ferroxidase that is considered to be an important source of copper in milk for normal neonatal development. We investigated the expression, subcellular localization and secretion of Cp in PMC42-LA cell culture models representative of resting, lactating and suckled human mammary epithelia. Both secreted Cp (sCp) and plasma membrane associated glycosylphosphatidylinositol-linked Cp (GPI-Cp) were expressed in PMC42-LA cells. In all three epithelial models (resting, lactating and suckled), the expression and secretion of copper-bound, ferroxidase active, Cp (holo-Cp) was dependent on media copper concentration. In low copper (bathocuproinedisulphonic acid/d-penicillamine treated models) there was greater than a 2-fold decrease in holo-Cp expression and secretion, which was mirrored by a 2-fold increase in the expression and secretion of copper-free Cp protein (apo-Cp). Cell surface biotinylation studies revealed that the state of PMC42-LA cell differentiation (functionality), and the level of extracellular copper, had no significant effect on the level of plasma membrane bound GPI-Cp. Quantitative real time PCR analyses determined that there was no significant (P > 0.05) difference in Cp mRNA levels across all copper conditions investigated (0, 5, 50 µM). However, there was a significant (P < 0.05) increase (∼2-fold) in Cp mRNA in both the lactating and suckled models in comparison to the resting model. Furthermore, the Cp mRNA increase in response to PMC42-LA differentiation corresponded with more secreted Cp protein, both apo and holo forms, indicating a link between function and Cp requirement. Our results provide significant insight on the regulation of Cp expression and secretion in lactation and copper incorporation into milk.

Zinc and infant nutrition.

Zinc is essential for a wide variety of cellular processes in all cells. It is a critical dietary nutrient, particularly in the early stages of life. In the early neonatal period, adequate sources of zinc can be obtained from breast milk. In rare circumstances, the mammary gland produces zinc deficient milk that is potentially lethal for exclusively breast-fed infants. This can be overcome by zinc supplementation to the infant. Alterations to key zinc transporters provide insights into the mechanisms of cellular zinc homeostasis. The bioavailability of zinc in food depends on the presence of constituents that may complex zinc. In many countries, zinc deficiency is a major health issue due to poor nourishment. Young children are particularly affected. Zinc deficiency can impair immune function and contributes to the global burden of infectious diseases including diarrhoea, pneumonia and malaria. Furthermore, zinc deficiency may extend its influence across generations by inducing epigenetic effects that alter the expression of genes. This review discusses the significance of adequate zinc nutrition in infants, factors that influence zinc nutrition, the consequences of zinc deficiency, including its contribution to the global burden of disease, and addresses some of the knowledge gaps in zinc biology.

Epigenetic Markers to Predict Conversion From Gestational Diabetes to Type 2 Diabetes.

CONTEXT: Lifestyle factors mediate epigenetic changes that can cause chronic diseases. Although animal and laboratory studies link epigenetic changes to diabetes, epigenetic information in women with gestational diabetes (GDM) and type 2 diabetes is lacking. OBJECTIVE: This study sought to measure epigenetic markers across pregnancy and early postpartum and identify markers that could be used as predictors for conversion from GDM to type 2 diabetes. DESIGN: Global histone H3 dimethylation was measured in white blood cells at three time points: 30 wk gestation, 8-10 wk postpartum, and 20 wk postpartum, from four groups of women with and without diabetes. SETTING AND PARTICIPANTS: A total of 39 participants (six to nine in each group) were recruited including: nondiabetic women; women with GDM who developed postpartum type 2 diabetes; women with GDM without postpartum type 2 diabetes; and women with type 2 diabetes. MAIN OUTCOME MEASURE: Percentages of dimethylation of H3 histones relative to total H3 histone methylation were compared between diabetic/nondiabetic groups using appropriate comparative statistics. RESULTS: H3K27 dimethylation was 50-60% lower at 8-10 and 20 wk postpartum in women with GDM who developed type 2 diabetes, compared with nondiabetic women. H3K4 dimethylation was 75% lower at 8-10 wk postpartum in women with GDM who subsequently developed type 2 diabetes compared with women who had GDM who did not. CONCLUSIONS: The percentage of dimethylation of histones H3K27 and H3K4 varied with diabetic state and has the potential as a predictive tool to identify women who will convert from GDM to type 2 diabetes.

Identification and topographical characterisation of microbial nanowires in Nostoc punctiforme.

Extracellular pili-like structures (PLS) produced by cyanobacteria have been poorly explored. We have done detailed topographical and electrical characterisation of PLS in Nostoc punctiforme PCC 73120 using transmission electron microscopy (TEM) and conductive atomic force microscopy (CAFM). TEM analysis showed that N. punctiforme produces two separate types of PLS differing in their length and diameter. The first type of PLS are 6-7.5 nm in diameter and 0.5-2 µm in length (short/thin PLS) while the second type of PLS are ~20-40 nm in diameter and more than 10 µm long (long/thick PLS). This is the first study to report long/thick PLS in N. punctiforme. Electrical characterisation of these two different PLS by CAFM showed that both are electrically conductive and can act as microbial nanowires. This is the first report to show two distinct PLS and also identifies microbial nanowires in N. punctiforme. This study paves the way for more detailed investigation of N. punctiforme nanowires and their potential role in cell physiology and symbiosis with plants.

The ZntA-like NpunR4017 plays a key role in maintaining homeostatic levels of zinc in Nostoc punctiforme.

Analysis of cellular response to zinc exposure provides insights into how organisms maintain homeostatic levels of zinc that are essential, while avoiding potentially toxic cytosolic levels. Using the cyanobacterium Nostoc punctiforme as a model, qRT-PCR analyses established a profile of the changes in relative mRNA levels of the ZntA-like zinc efflux transporter NpunR4017 in response to extracellular zinc. In cells treated with 18 µM of zinc for 1 h, NpunR4017 mRNA levels increased by up to 1300 % above basal levels. The accumulation and retention of radiolabelled (65)Zn by NpunR4107-deficient and overexpressing strains were compared to wild-type levels. Disruption of NpunR4017 resulted in a significant increase in zinc accumulation up to 24 % greater than the wild type, while cells overexpressing NpunR4107 accumulated 22 % less than the wild type. Accumulation of (65)Zn in ZntA(-) Escherichia coli overexpressing NpunR4017 was reduced by up to 21 %, indicating the capacity for NpunR4017 to compensate for the loss of ZntA. These findings establish the newly identified NpunR4017 as a zinc efflux transporter and a key transporter for maintaining zinc homeostasis in N. punctiforme.

Characterization of two cation diffusion facilitators NpunF0707 and NpunF1794 in Nostoc punctiforme.

AIMS: To characterize genes involved in maintaining homeostatic levels of zinc in the cyanobacterium Nostoc punctiforme. METHODS AND RESULTS: Metal efflux transporters play a central role in maintaining homeostatic levels of trace elements such as zinc. Sequence analyses of the N. punctiforme genome identified two potential cation diffusion facilitator (CDF) metal efflux transporters, Npun_F0707 (Cdf31) and Npun_F1794 (Cdf33). Deletion of either Cdf31or Cdf33 resulted in increased zinc retention over 3 h. Interestingly, Cdf31(-) and Cdf33(-) mutants showed no change in sensitivity to zinc exposure in comparison with the wild type, suggesting some compensatory capacity for the loss of each other. Using qRT-PCR, a possible interaction was observed between the two cdf's, where the Cdf31(-) mutant had a more profound effect on cdf33 expression than Cdf33(-) did on cdf31. Over-expression of Cdf31 and Cdf33 in ZntA(-) - and ZitB(-) -deficient Escherichia coli revealed function similarities between the ZntA and ZitB of E. coli and the cyanobacterial transporters. CONCLUSIONS: The data presented shed light on the function of two important transporters that regulate zinc homeostasis in N. punctiforme. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows for the first time the functional characterization of two cyanobacterial zinc efflux proteins belonging to the CDF family.