Sampling of riverine litter with citizen scientists--findings and recommendations.

The quantity and composition of litter at riversides and in the surface waters, as well as the occurrence of illegal dumping sites, were studied along four rivers in Chile. Data generated by volunteers were compared to the results from a professional survey, using an identical protocol. Litter was found in considerable quantities at the riversides and in the surface waters at all the sites investigated. A generalized linear mixed model analysis showed that the recorded litter densities did not differ between volunteers and professionals, even after controlling for river, site, or distance between sampling locations, demonstrating that the volunteers successfully applied the sampling protocol. Differences occurred with respect to litter composition, which is most likely due to difficulties in the classification of litter items and particles and to the underestimation of litter present in surface water samples. Even though this study was only conducted at a small number of rivers and sites, a comparatively consistent pattern of direct and intentional litter deposition at riversides was recorded, highlighting that river basins require more protection. The results also show that the citizen science approach can be a suitable means for more extensive litter surveys at riversides and in other natural environments.

Rivers as a source of marine litter--a study from the SE Pacific.

Composition and abundance of persistent buoyant litter (plastics, polystyrene and manufactured wood) were investigated at riversides and on adjacent coastal beaches of four rivers flowing into the SE Pacific Ocean. Persistent buoyant litter made up the main share of litter at riversides (36-82%) and on coastal beaches near the river mouths (67-86%). The characteristic litter composition of each river is attributable to human influences along its course. Riverine litter items were deposited to both sides of the river mouths on coastal beaches, and their abundance generally declined with distance from the river mouth. However, maximum litter accumulations were often found on beaches north of the river mouth, suggesting a long-term influence of the prevailing equatorward low-level jet along the Chilean coast. The results confirm that riverine transport has an important impact on litter abundances on coastal beaches.

Control of development and valepotriate production by auxins in micropropagated Valeriana glechomifolia.

Valeriana glechomifolia is a plant species endemic to southern Brazil that accumulates valepotriates, which are terpene derivatives, in all of its organs. Valepotriates are the presumed sedative generic components of the pharmaceutically used species of Valeriana. The influence of various concentrations of the auxins indole-3-acetic acid, indole-3-butyric acid and alpha-naphthaleneacetic acid on the growth of micropropagated V. glechomifolia was investigated under conditions of transient and continuous exposure. Changes in the development of roots and shoots as well as the production of the valepotriates acevaltrate, valtrate and didrovaltrate (analyzed by high-performance liquid chromatography) were evaluated. The best performance in valepotriate production, growth and survival under ex vitro conditions following plant acclimatization was achieved in the continuous presence of 5.71 microM IAA. When cultured in medium containing IAA plants produced stable levels of valepotriates throughout the entire cultivation period.

Functional dissection of the molybdate-responsive transcription regulator, ModE, from Escherichia coli.

The product of the Escherichia coli modE gene, ModE, is a member of a unique class of molybdate-responsive DNA binding proteins. Here we investigated the roles of the N- and C-terminal domains of ModE in mediating DNA binding and protein dimerization, respectively. Compared to the full-length protein, the N-terminal half of ModE has a greatly diminished capacity to bind the modA promoter in vitro and to repress expression from a modA-lacZ operon fusion in vivo. Fusing a protein dimerization domain, encoded by the C terminus of lambda CI repressor protein, to the truncated ModE protein generated a ModE-CI fusion protein that not only displayed a greatly increased in vivo repressor activity but could also substitute for ModE at the moaA and dmsA promoters. In the reciprocal experiment, we restored repressor activity to a truncated CI protein by addition of the C-terminal domain of ModE, which is comprised of two MopI-like subdomains. By an in vivo competition assay, we also demonstrated that the CI-ModE chimeric protein retained the ability to interact with wild-type ModE. Finally, specific deletions within the ModE portion of the CI-ModE protein chimera abolished both in vivo repression and the ability to interact with wild-type ModE. Together, these data demonstrate that the N-terminal domain of ModE is sufficient to mediate DNA binding, although efficient binding requires that ModE form a dimer, a function that is supplied by the C-terminal MopI-like subdomains.

Purification and characterization of the selenate reductase from Thauera selenatis.

Thauera selenatis is one of two isolated bacterial species that can obtain energy by respiring anaerobically with selenate as the terminal electron acceptor. The reduction of selenate to selenite is catalyzed by a selenate reductase, previously shown to be located in the periplasmic space of the cell. This study describes the purification of the enzyme from T. selenatis grown anaerobically with selenate. The enzyme is a trimeric alphabetagamma complex with an apparent Mr of 180,000. The alpha, beta, and gamma subunits are 96 kDa, 40 kDa, and 23 kDa, respectively, in size. The selenate reductase contains molybdenum, iron, and acid-labile sulfur as prosthetic group constituents. UV-visible absorption spectroscopy also revealed the presence of one cytochrome b per alphabetagamma complex. The Km for selenate was determined to be 16 microM, and the Vmax was 40 micromol/min/mg of protein. The enzyme is specific for the reduction of selenate; nitrate, nitrite, chlorate, and sulfate were not reduced at detectable rates. These studies constitute the first description of a selenate reductase, which represents a new class of enzymes. The significance of this enzyme in relation to cell growth and energy generation is discussed.

Characterization of the ModE DNA-binding sites in the control regions of modABCD and moaABCDE of Escherichia coli.

The Escherichia coli molybdate transporter, encoded by the modABCD operon, is negatively regulated by the modE gene product in response to the intracellular molybdate concentration. Utilizing an in vivo titration assay, we localized the ModE-binding site to the start of modA transcription. This localization was further characterized using in vitro gel-shift assays and DNase I footprinting. ModE bound the wild-type modA promoter with an apparent dissociation constant (Kd) of 45 nM, and addition of molybdate, in physiologically relevant amounts, significantly increased DNA binding. Consistent with these data, modA promoter fragments containing mutations that reduced ModE repression in vivo displayed proportionately higher apparent Kd values in vitro. DNase I footprinting of the modA promoter revealed a single protected region that overlapped the start site of transcription and extended from position -18 to +10, relative to the transcript start site. Gel-shifting assays, employing the promoter regions from the tor, nrf, moa and moe operons, revealed that ModE bound only the moa promoter region, with an apparent Kd of 24nM. Footprint analysis of the moaA promoter revealed a single protected region located immediately upstream of the putative -35 consensus sequence and extending from position -202 to -174, relative to the start of translation. In vivo expression of a moaA-lacZ operon fusion was stimulated twofold by ModE. However, relative to modA, binding of ModE to the moaA promoter appeared to be largely molybdate independent both in vitro and in vivo. These findings demonstrate that ModE acts both as a repressor and activator of the mod and moa operons, respectively, depending on the properties of the binding site.

Properties of the periplasmic ModA molybdate-binding protein of Escherichia coli.

The modABCD operon, located at 17 min on the Escherichia coli chromosome, encodes the protein components of a high affinity molybdate uptake system. Sequence analysis of the modA gene (GenBank L34009) predicts that it encodes a periplasmic binding protein based on the presence of a leader-like sequence at its N terminus. To examine the properties of the ModA protein, the modA structural gene was overexpressed, and its product was purified. The ModA protein was localized to the periplasmic space of the cell, and it was released following a gentle osmotic shock. The N-terminal sequence of ModA confirmed that a leader region of 24 amino acids was removed upon export from the cell. The apparent size of ModA is 31.6 kDa as determined by gel sieve chromatography, whereas it is 22.5 kDa when examined by SDS-polyacrylamide gel electrophoresis. A ligand-dependent protein mobility shift assay was devised using a native polyacrylamide gel electrophoresis protocol to examine binding of molybdate and other anions to the ModA periplasmic protein. Whereas molybdate and tungstate were bound with high affinity (approximately 5 microM), sulfate, chromate, selenate, phosphate, and chlorate did not bind even when tested at 2 mM. A UV spectral assay revealed apparent Kd values of binding for molybdate and tungstate of 3 and 7 microM, respectively. Strains defective in the modA gene were unable to transport molybdate unless high levels of the anion were supplied in the medium. Therefore the modA gene product is essential for high affinity molybdate uptake by the cell. Tungstate interference of molybdate acquisition by the cell is apparently due in part to the high affinity of the ModA protein for this anion.

Regulation of the molybdate transport operon, modABCD, of Escherichia coli in response to molybdate availability.

The mod (chlD) locus at 17 min on the Escherichia coli chromosome encodes a high-affinity molybdate uptake system. To further investigate the structure and regulation of these genes, the DNA region upstream of the previously identified modBC (chlJD) genes was cloned and sequenced. A single open reading frame, designated modA, was identified and appears to encode a periplasmic binding protein for the molybdate uptake system. To determine how the mod genes are regulated in response to molybdate, nitrate, and oxygen, we constructed a series of mod-lacZ operon fusions to the upstream region and introduced them in single copy onto the E. coli chromosome. Whereas molybdate limitation resulted in elevated mod-lacZ expression, neither oxygen nor nitrate had any significant effect on gene expression. A regulatory motif, CATAA, located at the modA promoter was identified and shown to be required for molybdate-dependent control of the modABCD operon. Mutations within this sequence resulted in nearly complete derepression of gene expression and suggest that transcription of the operon is mediated by a molybdenum-responsive regulatory protein.

Thauera selenatis gen. nov., sp. nov., a member of the beta subclass of Proteobacteria with a novel type of anaerobic respiration.

A recently isolated, selenate-respiring microorganism (strain AXT [T = type strain]) was classified by using a polyphasic approach in which both genotypic and phenotypic characteristics were determined. Strain AXT is a motile, gram-negative, rod-shaped organism with a single polar flagellum. On the basis of phenotypic characteristics, this organism can be classified as a Pseudomonas sp. However, a comparison of the 16S rRNA sequence of strain AXT with the sequences of other organisms indicated that strain AXT is most similar to members of the beta subclass (level of similarity, 86.8%) rather than to members of the gamma subclass (level of similarity, 80.2%) of the Proteobacteria. The presence of the specific polyamine 2-hydroxyputrescine and the presence of a ubiquinone with eight isoprenoid units in the side chain (ubiquinone Q-8) excluded strain AXT from the authentic genus Pseudomonas and allowed placement in the beta subclass of the Proteobacteria. Within the beta subclass, strain AXT is related to Iodobacter fluvatile. The phylogenetic distance (level of similarity, less than 90%), as well as a lack of common phenotypic characteristics between these organisms, prevents classification of strain AXT as a member of the genus Iodobacter. In addition, strain AXT possesses a unique mechanism for anaerobic respiration, which allows it to utilize selenate as an electron acceptor without interference by nitrate. Therefore, we propose that strain AXT should be the first member of a new genus and species, Thauera selenatis.

The terminal reductases for selenate and nitrate respiration in Thauera selenatis are two distinct enzymes.

A number of approaches have been used to show that a recently isolated selenate-respiring bacterium, Thauera selenatis, is able to synthesize both a selenate reductase (SR) and a nitrate reductase (NR). (i) The pH optimum of the SR was found to be 6.0; that of the NR was 7.0. (ii) The presence of nitrate did not inhibit selenate reduction in selenate-grown cells. (iii) In cell extracts, the highest SR or NR activity was observed in cells grown with the respective electron acceptor. (iv) Mutants that were unable to grow with nitrate as the terminal electron acceptor and lacked NR activity were isolated; these mutants grew normally with selenate and synthesized SR. (v) The SR was found in the periplasmic space of the cell, whereas the NR was present in the cytoplasmic membrane. A hypothetical electron transport system involving the SR is described.

Complementation of the Saccharomyces cerevisiae srb1-1 mutation: an autoselection system for stable plasmid maintenance.

The autonomously replicating plasmid YEpSS1, containing the S. cerevisiae SOD1 and SRB1 genes, was highly unstable in a wild-type strain. When transformed into a fragile srb1-1 mutant host, the same plasmid displayed different characteristics depending on the growth medium used. Both batch and continuous culture experiments demonstrated that the plasmid was very unstable when the transformed strain SLU15 was grown in the presence of an osmotic stabiliser (10% w/v sorbitol). However, in the absence of the osmoticum, nearly 100% of the cells retained the plasmid and produced the Sod1 protein after 80 generations of growth.