Fibers spreading worldwide: Microplastics and other anthropogenic litter in an Arctic freshwater lake.

We investigated the presence of microplastics and other anthropogenic litter in the sediments adhered to rocks of an Arctic freshwater lake at Ny-Ålesund (Svalbard Archipelago, 78°N; 11°E). Most of the sampled microparticles were fibers (>90%). The identification of polymer types and additives was performed by combining three spectroscopic techniques, namely Raman Microscopy, Fourier-Transform Infrared microspectroscopy (µFTIR) and Synchrotron Radiation µFTIR (SR-FTIR). SR-FTIR confirmed the presence of poly(ethylene terephthalate) fibers, while RAMAN spectroscopy provided evidence of fibers containing industrial additives. Our results estimated an average concentration of 400 microparticles/m2 of rocks identified as anthropogenic litter, which included an estimation of 90 microplastics/m2 identified as polyester fibers; the rest are mostly natural fibers with evidence of anthropogenic origin. Taken together, the results proved the occurrence of anthropogenic pollutants in remote polar areas. Their probable origin is the long range atmospheric transport.

Two novel cyanobacterial bioluminescent whole-cell bioreporters based on superoxide dismutases MnSod and FeSod to detect superoxide anion.

This work describes the construction of two novel self-luminescent bioreporter strains of the cyanobacterium Nostoc sp. PCC 7120 by fusing the promoter region of the sodA and sodB genes (encoding the superoxide dismutases MnSod and FeSod, respectively) to luxCDABE from Photorhabdus luminescens aimed at detecting pollutants that generate reactive oxygen species (ROS), particularly O2-. Bioreporters were tested against methyl viologen (MV) as the inducer of superoxide anion (O2-). Both bioreporters were specific for O2- and Limits of detection (LODs) and Maximum Permissive Concentrations (MPCs) were calculated: Nostoc sp. PCC 7120 pBG2154 (sodA) had a range of detection from 400 to 1000 pM of MV and for Nostoc sp. PCC 7120 pBG2165 (sodB) the range of detection was from 500 to 1800 pM of MV after 5 h-exposure. To further validate the bioreporters, they were tested with the emerging pollutant Triclosan which induced bioluminescence in both strains. Furthermore, the bioreporters performance was tested in two real environmental samples with different water matrix complexity, spiked with MV. Both bioreporters were induced by O2- in these environmental samples. In the case of the river water sample, the amount of bioavailable MV as calculated from the bioreporters output was similar to that nominally added. For the waste water sample, the bioavailable MV concentration detected by the bioreporters was one order of magnitude lower than nominal. These differences could be due to MV complexation with organic matter and/or co-occurring organic contaminants. These results confirm their high sensitivity to O2- and their suitability to detect oxidative stress-generating pollutants in fresh-waters.

Defining an additivity framework for mixture research in inducible whole-cell biosensors.

A novel additivity framework for mixture effect modelling in the context of whole cell inducible biosensors has been mathematically developed and implemented in R. The proposed method is a multivariate extension of the effective dose (EDp) concept. Specifically, the extension accounts for differential maximal effects among analytes and response inhibition beyond the maximum permissive concentrations. This allows a multivariate extension of Loewe additivity, enabling direct application in a biphasic dose-response framework. The proposed additivity definition was validated, and its applicability illustrated by studying the response of the cyanobacterial biosensor Synechococcus elongatus PCC 7942 pBG2120 to binary mixtures of Zn, Cu, Cd, Ag, Co and Hg. The novel method allowed by the first time to model complete dose-response profiles of an inducible whole cell biosensor to mixtures. In addition, the approach also allowed identification and quantification of departures from additivity (interactions) among analytes. The biosensor was found to respond in a near additive way to heavy metal mixtures except when Hg, Co and Ag were present, in which case strong interactions occurred. The method is a useful contribution for the whole cell biosensors discipline and related areas allowing to perform appropriate assessment of mixture effects in non-monotonic dose-response frameworks.

Expression of luxCD-E in Anabaena sp. can replace the use of exogenous aldehyde for in vivo localization of transcription by luxAB.

The genes luxCDABE from four luminescent bacteria suffice for light production in Escherichia coli [Meighen, Microbiol. Rev. 55 (1991) 123-142]. We have inserted these gene clusters between inverted polylinkers, and placed the resulting cassettes as reporters within derivatives of transposon Tn5. Anabaena sp. strain PCC 7120 was mutagenized with these transposons. The luminescence of all but the most highly self-luminescent resulting derivatives of Anabaena sp. was strongly dependent on exogenously added aldehyde. Thus, luminescence based on luxCDABE is multiplicatively limited by production of luciferase and aldehyde. No toxicity was observed over protracted periods of luminescence. By deletion, new cassettes were derived in which only the aldehyde biosynthetic genes, luxCD-E, remained intact. Transcription was localized at the single-cell level in strains of cyanobacteria bearing constitutively expressed Xenorhabdus luminescens luxCD-E on a plasmid and relatively weakly expressed, developmentally regulated luxAB from Vibrio spp. in the chromosome. The developmentally critical gene, hetR, was thereby shown to remain active in mature heterocysts.

Role of external calcium in homeostasis of intracellular pH in the cyanobacterium Anabaena sp. strain PCC7120 exposed to low pH.

The role of external Ca2+ in the homeostasis of intracellular pH (pHi) of Anabaena sp. strain PCC7120 in response to a decrease in the external pH (pHex) has been studied in cell suspensions. Increase in cytoplasmic pH after acid shock is dependent on the presence of Ca2+ in the medium. The observed Ca2+ -mediated alkalization of the cytoplasm depends on the extent of the shift in external pH. Acid pH shifts resulted in an increased permeability of the cytoplasmic membrane to protons, which could be reversed by increasing the concentration of Ca2+ in the medium. Thus, the ability of Ca2+ to increase cytoplasmic pH might be correlated with an inhibition of net proton uptake by increasing concentrations of external Ca2+ under these conditions. This combined response resulted in the generation and maintenance of a larger pH gradient (ΔpH) at acid external pH values. All Ca2+ channel blockers tested, such as verapamil and LaCl3 , inhibited the observed Ca2+ -mediated response. On the other hand, the Ca ionophore calcimycin (compound A23187) was agonistic, and stimulated both cytoplasmic alkalization and inhibition of net proton uptake. The protonophorous uncoupler carbonylcyanide m-chlorophenyl hydrazone, inhibited this Ca2+ -mediated response, whereas monensin, an inhibitor of the Na+ /H+ antiporter, had no significant effect. The results of the present study suggest that an influx of Ca2+ from the extracellular space is required for the regulation of cytoplasmic pH in Anabaena sp. strain PCC7120 exposed to low external pH values.

mrpA, a gene with roles in resistance to Na+ and adaptation to alkaline pH in the cyanobacterium Anabaena sp. PCC7120.

Transposon mutagenesis of Anabaena sp. PCC7120 led to the isolation of a mutant strain, PHB11, which grew poorly at pH values above 10. The mutant strain exhibited pronounced Na+ sensitivity; this sensitivity was higher under basic conditions. Mutant PHB11 also showed an inhibition of photosynthesis that was much more pronounced at alkaline pH. Reconstruction of the transposon mutation of PHB11 in the wild-type strain reproduced the phenotype of the original mutant. The wild-type version of the mutated gene was cloned and the mutation complemented. In mutant strain PHB11, the transposon had inserted within an ORF that is part of a seven-ORF operon with significant sequence similarity to a family of bacterial operons that are believed to code for a novel multiprotein cation/proton antiporter primarily involved in resistance to salt stress and adaptation to alkaline pH. The Anabaena operon was denoted mrp (multiple resistance and pH adaptation) following the nomenclature of the Bacillus subtilis operon; the ORF mutated in PHB11 corresponded to mrpA. Computer analysis suggested that all seven predicted Anabaena Mrp proteins were highly hydrophobic with several transmembrane domains; in fact, the predicted protein sequences encoded by mrpA, mrpB and mrpC showed significant similarity to hydrophobic subunits of the proton pumping NADH : ubiquinone oxidoreductase. In vivo expression studies indicated that mrpA is induced with increasing external Na+ concentrations and alkaline pH; mrpA is also upregulated under inorganic carbon (Ci) limitation. The biological significance of a putative cyanobacterial Mrp complex is discussed.

A third genetic locus required for the formation of heterocysts in Anabaena sp. strain PCC 7120.

Mutagenesis of Anabaena sp. strain PCC 7120 with a derivative of transposon Tn5 led to the isolation of a mutant strain, P6, in which heterocysts are not formed (A. Ernst, T. Black, Y. Cai, J.-M. Panoff, D. N. Tiwari, and C. P. Wolk, J. Bacteriol. 174:6025-6032, 1992). Reconstruction of the transposon mutation of P6 in the wild-type strain reproduced the phenotype of the original mutant. Analysis by pulsed-field gel electrophoresis localized the transposition at ca. 3.44 Mb on the physical map of the chromosome of wild-type Anabaena sp. The transposon was situated within an open reading frame (ORF), which we denote hetP, whose wild-type form was cloned and also sequenced. The predicted HetP protein was not found to show significant sequence similarity to other proteins. The mutation in strain P6 could be complemented by a clone of a fragment of wild-type DNA that includes hetP and at least one additional ORF 3' from hetP, but not by a clone that includes hetP as its only ORF. The latter clone proved highly toxic. The phenotype of the P6 mutant may, therefore, be due to a polar effect of the insertion of the transposon. Filaments of strain P6 and of the wild-type strain, when bearing the complementing fragment on a pDU1-based plasmid, showed an increased frequency of clustered heterocysts compared with that of the wild-type strain.

Two mutations that block heterocyst differentiation have different effects on akinete differentiation in Nostoc ellipsosporum.

Evident differentiation of vegetative cells into heterocysts in Anabaena sp. strain PCC 7120 is prevented by insertions in genes hetR and hetP. Nostoc ellipsosporum possesses single copies of genes that hybridize with hetR and hetP. In mutant NE2 of N. ellipsosporum, in which hetR is interrupted by an insert, and in a double recombinant of wild-type N. ellipsosporum with a plasmid that bears an interrupted copy of hetR, neither heterocysts nor akinetes are formed. When an intact copy of hetR from Anabaena sp. strain PCC 7120 was added to NE2 the ability to form both heterocysts and akinetes was restored. In contrast to the hetR mutant, a hetP mutant of N. ellipsosporum could form akinetes, but heterocyst formation was blocked. Use of luxAB, encoding luciferase, as a reporter, and use of luxC, luxD and luxE to generate aldehyde (a substrate for the luciferase reaction), permitted visualization of the expression of hetR at the level of single cells; hetR was expressed in akinetes.

Binding of cadmium by cyanobacterial growth media: free ion concentration as a toxicity index to the cyanobacterium Nostoc UAM 208.

A quantitative study of cadmium binding to three different growth media for nitrogen-fixing cyanobacteria was done with the aid of a solid state ion-specific electrode. Kratz and Myers modified medium and Arnon medium bound large amounts of Cd2+, BG11o medium had less binding capacity. Of the media components, phosphate ion showed the greatest ability to bind Cd2+. Different pHs, the size of cell inoculum and two buffers (Tricine and HEPES, 25 mM) also changed the availability of free cadmium ion in solution. The effect of free Cd2+ ion towards the cyanobacterium Nostoc UAM 208, isolated from a heavy metal polluted environment, also was tested. The effective concentration affecting 50% of population (EC50), at 120 h of exposure, was less for nitrogenase activity (0.26 microgram/mL) than for growth (0.55 microgram/mL), suggesting that this enzyme activity is more sensitive to cadmium than growth. Furthermore, cadmium toxicity was influenced by the addition of buffers to the growth medium. In the presence of buffer, Tricine (25 mM), growth and nitrogenase activity was reduced by 50% at a total cadmium concentration of about 115 micrograms/mL, although no free ion was detected in this case. These results suggest that although generally cadmium toxicity is a function of free metal ion concentration, this can also vary in the presence of complexing agents.

A calcium signal is involved in heterocyst differentiation in the cyanobacterium Anabaena sp. PCC7120.

The impact of calcium signals in virtually all cells has led to the study of their role in prokaryotic organisms as stress response modulators. Cell differentiation in adverse conditions is a common Ca(2+)-requiring response. Nitrogen starvation induces the differentiation of N(2)-fixing heterocysts in the filamentous cyanobacterium Anabaena sp. PCC7120. This paper reports the use of a recombinant strain of this organism expressing the photoprotein aequorin to monitor the intracellular free-calcium concentration during the course of heterocyst differentiation. A specific calcium signature that is triggered exclusively when cells are deprived of combined nitrogen and generated by intracellular calcium stores was identified. The intracellular calcium signal was manipulated by treatment with specific calcium drugs, and the effect of such manipulation on the process of heterocyst differentiation was subsequently assessed. Suppression, magnification or poor regulation of this signal prevented the process of heterocyst differentiation, thereby suggesting that a calcium signal with a defined set of kinetic parameters may be required for differentiation. A hetR mutant of Anabaena sp. PCC7120 that cannot differentiate into heterocysts retains, however, the capacity to generate the calcium transient in response to nitrogen deprivation, strongly suggesting that Ca(2+) may be involved in a very early step of the differentiation process.

Use of recombinant aequorin to study calcium homeostasis and monitor calcium transients in response to heat and cold shock in cyanobacteria.

We investigated the possibility of Ca(2+) signaling in cyanobacteria (blue-green algae) by measuring intracellular free Ca(2+) levels ([Ca(2+)](i)) in a recombinant strain of the nitrogen fixing cyanobacterium Anabaena strain sp. PCC7120, which constitutively expresses the Ca(2+)-binding photoprotein apoaequorin. The homeostasis of intracellular Ca(2+) in response to increasing external Ca(2+) has been studied in this strain. The resting level of free Ca(2+) in Anabaena was found to be between 100 and 200 nM. Additions of increasing concentrations of external Ca(2+) gave a transient burst of [Ca(2+)](i) followed by a very quick decline, reaching a plateau within seconds that brought the level of [Ca(2+)](i) back to the resting value. These results indicate that Anabaena strain sp. PCC7120 is able to regulate its internal Ca(2+) levels. We also monitored Ca(2+) transients in our recombinant strain in response to heat and cold shock. The cell's response to both stresses was dependent on the way they were induced. The use of inhibitors suggests that heat shock mobilizes cytosolic Ca(2+) from both intracellular and extracellular sources, while the Ca(2+) source for cold shock signaling is mostly extracellular.

A transposition-induced mutant of Nostoc ellipsosporum implicates an arginine-biosynthetic gene in the formation of cyanophycin granules and of functional heterocysts and akinetes.

In strain NE1 of Tn5-1058-mutagenized Nostoc ellipsosporum, the transposon was found within a gene whose translation product is similar in amino acid sequence to the arginine-biosynthetic protein N-acetylglutamate semialdehyde dehydrogenase encoded by argC of Bacillus subtilis. The argC reported from Anabaena sp. strain PCC 7120 hybridized to a sequence different from the one interrupted by the transposon in NE1. The newly identified gene from N. ellipsosporum was denoted argL. The argL mutation renders certain processes in strain NE1 conditionally dependent on provision of L-arginine. Heterocysts and apparent akinetes that formed in the absence of added L-arginine failed to fix dinitrogen or to germinate, respectively, and lacked granules of cyanophycin, composed of copolymers of arginine and aspartic acid. However, apparent akinetes that differentiated upon growth of the mutant in the presence of L-arginine plus nitrate formed cyanophycin granules and could regenerate a new culture.

Characterization of devA, a gene required for the maturation of proheterocysts in the cyanobacterium Anabaena sp. strain PCC 7120.

Mutant M7, obtained by transposon mutagenesis of the cyanobacterium Anabaena sp. strain PCC 7120, is impaired in the development of mature heterocysts. Under aerobic conditions, the mutant is unable to fix N2 because of a deficiency of at least two components of the oxygen-protective mechanisms: a hemoprotein-coupled oxidative reaction and heterocyst-specific glycolipids. DNA contiguous with the inserted transposon was recovered from the mutant and sequenced. The transposon had inserted itself within a 732-bp open reading frame designated devA. The wild-type form of devA, obtained from a lambda-EMBL3 library of Anabaena sp. DNA, had the identical sequence. Directed mutagenesis of devA in the wild-type strain showed that the phenotype of the mutant was caused by insertion of the transposon. The wild-type form of devA on a shuttle vector complemented the mutation in M7. Expression of devA by whole filaments, monitored following nitrogen stepdown by using luxAB as the reporter, increased ca. eightfold during differentiation; the increase within differentiating cells was much greater. The deduced sequence of the DevA protein shows strong similarity to the ATP-binding subunit of binding protein-dependent transport systems. The product of devA may, therefore, be a component of a periplasmic permease that is required for the transition from a proheterocyst to a mature, nitrogen-fixing heterocyst.

pbpB, a gene coding for a putative penicillin-binding protein, is required for aerobic nitrogen fixation in the cyanobacterium Anabaena sp. strain PCC7120.

Transposon mutagenesis of Anabaena sp. strain PCC7120 led to the isolation of a mutant strain, SNa1, which is unable to fix nitrogen aerobically but is perfectly able to grow with combined nitrogen (i. e., nitrate). Reconstruction of the transposon mutation of SNa1 in the wild-type strain reproduced the phenotype of the original mutant. The transposon had inserted within an open reading frame whose translation product shows significant homology with a family of proteins known as high-molecular-weight penicillin-binding proteins (PBPs), which are involved in the synthesis of the peptidoglycan layer of the cell wall. A sequence similarity search allowed us to identify at least 12 putative PBPs in the recently sequenced Anabaena sp. strain PCC7120 genome, which we have named and organized according to predicted molecular size and the Escherichia coli nomenclature for PBPs; based on this nomenclature, we have denoted the gene interrupted in SNal as pbpB and its product as PBP2. The wild-type form of pbpB on a shuttle vector successfully complemented the mutation in SNa1. In vivo expression studies indicated that PBP2 is probably present when both sources of nitrogen, nitrate and N(2), are used. When nitrate is used, the function of PBP2 either is dispensable or may be substituted by other PBPs; however, under nitrogen deprivation, where the differentiation of the heterocyst takes place, the role of PBP2 in the formation and/or maintenance of the peptidoglycan layer is essential.