Francesco Pomati

Biosynthetic Intermediate Analysis and Functional Homology Reveal a Saxitoxin Gene Cluster in Cyanobacteria

ABSTRACT Saxitoxin (STX) and its analogues cause the paralytic shellfish poisoning (PSP) syndrome, which afflicts human health and impacts coastal shellfish economies worldwide. PSP toxins are unique alkaloids, being produced by both prokaryotes and eukaryotes. Here we describe a candidate PSP toxin biosynthesis gene cluster (sxt) from Cylindrospermopsis raciborskii T3. The saxitoxin biosynthetic pathway is encoded by more than 35 kb, and comparative sequence analysis assigns 30 catalytic functions to 26 proteins. STX biosynthesis is initiated with arginine, S-adenosylmethionine, and acetate by a new type of polyketide synthase, which can putatively perform a methylation of acetate, and a Claisen condensation reaction between propionate and arginine. Further steps involve enzymes catalyzing three heterocyclizations and various tailoring reactions that result in the numerous isoforms of saxitoxin. In the absence of a gene transfer system in these microorganisms, we have revised the description of the known STX biosynthetic pathway, with in silico functional inferences based on sxt open reading frames combined with liquid chromatography-tandem mass spectrometry analysis of the biosynthetic intermediates. Our results indicate the evolutionary origin for the production of PSP toxins in an ancestral cyanobacterium with genetic contributions from diverse phylogenetic lineages of bacteria and provide a quantum addition to the catalytic collective available for future combinatorial biosyntheses. The distribution of these genes also supports the idea of the involvement of this gene cluster in STX production in various cyanobacteria.

THE FRESHWATER CYANOBACTERIUM PLANKTOTHRIX SP. FP1: MOLECULAR IDENTIFICATION AND DETECTION OF PARALYTIC SHELLFISH POISONING TOXINS

A filamentous cyanobacterium, belonging to the Order of Oscillatoriales, was found to be responsible for a toxic algal bloom in Lake Varese, Italy, during the summer of 1997. Morphological characters, as well as near complete 16S rRNA gene sequencing, revealed that the dominant species of the bloom was most closely related to the genus Planktothrix. In addition, genetic analysis of the phycocyanin operon of Planktothrix sp. FP1 revealed a novel primary structure, previously undescribed within the cyanobacteria, which was used as a genetic marker for rapid detection and identification of this toxic strain. The occurrence of saxitoxin (STX), a principal toxin in paralytic shellfish poisoning (PSP), was confirmed in the natural bloom sample by both pre‐column and post‐column derivatization high‐performance liquid chromatography (HPLC) analyses, and eventually by liquid chromatography/mass spectrometry (LC/MS). The toxicity of this field sample was also revealed by electrophysiological assays in which the extract inhibited 90% of the voltage‐dependent Na+ current in human neuroblastoma cells at the STX concentration of 80 nM. The cultured strain showed a lower physiologic activity than the bloom sample (67% blockage of Na+ current at a toxin concentration of 200 nM), and STX was detected only by pre‐column HPLC, indicating the presence of a compound structurally close to STX. Chemical and molecular genetic analyses performed here add Planktothrix sp. FP1 to the growing list of diverse cyanobacterial species capable of synthesizing STX and its related compounds.

Biological Pump Control of the Fate and Distribution of Hydrophobic Organic Pollutants in Water and Plankton

The goal of this study was to experimentally assess the coupling between primary producer biomass dynamics and the distribution and fate of persistent organic pollutants (POPs) in a lake pelagic ecosystem. This was done by following the short-term evolution of polychlorinated biphenyl (PCB) concentrations in water and biota (phytoplankton and zooplankton) and the variability of bioconcentration (BCF), biomagnification (BMF), and bioaccumulation (BAF) factors during the development of a typical spring ecological progression in which the phytoplankton bloom is followed by a peak in the zooplankton abundance. The bulk of compounds with log K(OW) > 6.5 in the lake epilimnion was mainly associated with primary producer biomass. The phytoplankton biological pump was a major driver of POP export from the epilimnion, causing the decline of dissolved-phase concentrations. The BCF of phytoplankton for the more hydrophobic PCBs showed minima during the period of biomass climax. The concentration in the zooplankton of all selected PCBs sharply declined from March to May, with BAFs having minima in the post algal bloom phase. Biomagnification occurred during the pre algal bloom and algal bloom phases but appeared to be absent during the post algal bloom. This study highlights the occurrence of a prompt and complex response in the fate and distribution of POPs to dynamic biogeochemical control. Within the frame of the ecological succession, phytoplankton and zooplankton biomass dynamics produced bioaccumulation metrics varying over 1-2 orders of magnitude in the time frame of a few weeks and resulted in reduced trophic web exposure.

Detection of Saxitoxin-Producing Cyanobacteria and Anabaena circinalis in Environmental Water Blooms by Quantitative PCR

ABSTRACT Saxitoxins (STXs) are carbamate alkaloid neurotoxins produced by marine “red tide” dinoflagellates and several species of freshwater filamentous cyanobacteria, including Anabaena circinalis, Aphanizomenon spp., Lyngbya wollei, and Cylindrospermopsis raciborskii. A specific quantitative PCR (qPCR) method based on SYBR green chemistry was developed to quantify saxitoxin-producing Anabaena circinalis cyanobacteria, which are major bloom-forming freshwater cyanobacteria. The aim of this study was to infer the potential toxigenicity of samples by determining the copy number of a unique and unusual polyketide synthase (PKS) sequence (sxtA) in the STX biosynthesis gene cluster identified in cyanobacteria. Our qPCR approach was applied to water samples collected from different Australian lakes, dams, and rivers. The STX concentration and cyanobacterial cell density of these blooms were also determined by high-pressure liquid chromatography (HPLC) and microscopic cell counting, respectively. STX concentrations correlated positively with STX gene copy numbers, indicating that the latter can be used as a measure of potential toxigenicity in Anabaena circinalis and possibly other cyanobacterial blooms. The qPCR method targeting STX genes can also be employed for both monitoring and ecophysiological studies of toxic Anabaena circinalis blooms and potentially several other STX-producing cyanobacteria.

An Automated Platform for Phytoplankton Ecology and Aquatic Ecosystem Monitoring

High quality monitoring data are vital for tracking and understanding the causes of ecosystem change. We present a potentially powerful approach for phytoplankton and aquatic ecosystem monitoring, based on integration of scanning flow-cytometry for the characterization and counting of algal cells with multiparametric vertical water profiling. This approach affords high-frequency data on phytoplankton abundance, functional traits and diversity, coupled with the characterization of environmental conditions for growth over the vertical structure of a deep water body. Data from a pilot study revealed effects of an environmental disturbance event on the phytoplankton community in Lake Lugano (Switzerland), characterized by a reduction in cytometry-based functional diversity and by a period of cyanobacterial dominance. These changes were missed by traditional limnological methods, employed in parallel to high-frequency monitoring. Modeling of phytoplankton functional diversity revealed the importance of integrated spatiotemporal data, including circadian time-lags and variability over the water column, to understand the drivers of diversity and dynamic processes. The approach described represents progress toward an automated and trait-based analysis of phytoplankton natural communities. Streamlining of high-frequency measurements may represent a resource for understanding, modeling and managing aquatic ecosystems under impact of environmental change, yielding insight into processes governing phytoplankton community resistance and resilience.

The toxicity of chemical pollutants in dynamic natural systems: the challenge of integrating environmental factors and biological complexity.

The dynamics of abiotic and biotic environmental factors, like temperature and predation, can strongly influence the effects of anthropogenic chemical pollutants in natural systems. Responses to toxicants and their interactions with environmental factors can occur at varying temporal scales and at different levels of biological complexity (from cells to organisms, populations, communities and ecosystems). Environmental factors may affect tolerance to toxic pollutants under non-stressful conditions, and cause adverse multiple stressor effects under stressful conditions. Adaptive processes, however, have the potential to either mitigate (by co-tolerance) or increase (due to associated costs) the sensitivity of individuals, populations, and communities to pollutants through selection and evolution of traits (at the individual and population levels) and changes in species composition (at the community level). Responses to such multiple stressor effects on different biological levels and temporal scales are not considered in current risk assessment practices. We suggest that these effects should and can be addressed by: (i) designing ecotoxicological experiments with temporal exposure patterns that accommodate adaptive processes, (ii) using trait-based approaches to assess biological responses and natural selection in an integrated manner, and (iii) using energy allocation models to link responses at different levels of biological organization.

Novel homologs of the multiple resistance regulator marA in antibiotic-contaminated environments

Antibiotics are commonly detected in the environment as contaminants. Exposure to antibiotics may induce antimicrobial-resistance, as well as the horizontal transfer of resistance genes in bacterial populations. We selected the resistance gene marA, mediating resistance to multiple antibiotics, and explored its distribution in sediment and water samples from surface and sewage treatment waters. Ciprofloxacin and ofloxacin (fluoroquinolones), sulphamethoxazole (sulphonamide), erythromycin, clarythromycin, and spiramycin (macrolides), lincomycin (lincosamide), and oxytetracycline (tetracycline) were measured in the same samples to determine antibiotic contamination. Bacterial populations from environmental samples were challenged with antibiotics to identify resistant isolates. The gene marA was found in almost all environmental samples and was confirmed by PCR amplification in antibiotic-resistant colonies. 16S rDNA sequencing revealed that the majority of resistant isolates belonged to the Gram-positive genus Bacillus, not previously known to possess the regulator marA. We assayed the incidence of marA in environmental bacterial populations of Escherichia coli and Bacillus by quantitative real-time PCR in correlation with the levels of antibiotics. Phylogenetic analysis indicated the possible lateral acquisition of marA by Bacillus from Gram-negative Enterobacteriaceae revealing a novel marA homolog in Bacillus. Quantitative PCR assays indicate that the frequency of this gene in antropised environments seems to be related to bacterial exposure to water-borne antibiotics.

Identification of an Na+-Dependent Transporter Associated with Saxitoxin-Producing Strains of the Cyanobacterium Anabaena circinalis

ABSTRACT Blooms of the freshwater cyanobacterium Anabaena circinalis are recognized as an important health risk worldwide due to the production of a range of toxins such as saxitoxin (STX) and its derivatives. In this study we used HIP1 octameric-palindrome repeated-sequence PCR to compare the genomic structure of phylogenetically similar Australian isolates of A. circinalis. STX-producing and nontoxic cyanobacterial strains showed different HIP1 (highly iterated octameric palindrome 1) DNA patterns, and characteristic interrepeat amplicons for each group were identified. Suppression subtractive hybridization (SSH) was performed using HIP1 PCR-generated libraries to further identify toxic-strain-specific genes. An STX-producing strain and a nontoxic strain of A. circinalis were chosen as testers in two distinct experiments. The two categories of SSH putative tester-specific sequences were characterized by different families of encoded proteins that may be representative of the differences in metabolism between STX-producing and nontoxic A. circinalis strains. DNA-microarray hybridization and genomic screening revealed a toxic-strain-specific HIP1 fragment coding for a putative Na+-dependent transporter. Analysis of this gene demonstrated analogy to the mrpF gene of Bacillus subtilis, whose encoded protein is involved in Na+-specific pH homeostasis. The application of this gene as a molecular probe in laboratory and environmental screening for STX-producing A. circinalis strains was demonstrated. The possible role of this putative Na+-dependent transporter in the toxic cyanobacterial phenotype is also discussed, in light of recent physiological studies of STX-producing cyanobacteria.

Networking our way to better ecosystem service provision

The ecosystem services (EcoS) concept is being used increasingly to attach values to natural systems and the multiple benefits they provide to human societies. Ecosystem processes or functions only become EcoS if they are shown to have social and/or economic value. This should assure an explicit connection between the natural and social sciences, but EcoS approaches have been criticized for retaining little natural science. Preserving the natural, ecological science context within EcoS research is challenging because the multiple disciplines involved have very different traditions and vocabularies (common-language challenge) and span many organizational levels and temporal and spatial scales (scale challenge) that define the relevant interacting entities (interaction challenge). We propose a network-based approach to transcend these discipline challenges and place the natural science context at the heart of EcoS research.

Effects of saxitoxin (STX) and veratridine on bacterial Na+ -K+ fluxes: a prokaryote-based STX bioassay.

ABSTRACT Saxitoxin (STX) is a potent natural sodium channel blocker and represents a significant health concern worldwide. We describe here the antagonistic effects of STX and veratridine (VTD), an Na+ channel activator, on three gram-negative bacteria and their application to an STX bioassay. STX reduced the total cellular levels of both Na+ and K+, as measured by flame photometry, whereas VTD increased the cellular concentrations relative to control ion fluxes in the cyanobacterium Cylindrospermopsis raciborskii AWT205. Endogenous STX production in toxic cyanobacterial strains of C. raciborskii and Anabaena circinalis prevented cell lysis induced by VTD stress. Microscopic cell counts showed that non-STX producing cyanobacteria displayed complete cell lysis and trichome fragmentation 5 to 8 h after addition of VTD and vanadate (VAN), an inhibitor of sodium pumps. The addition of STX, or its analogue neoSTX, prior to treatment with VTD plus VAN prevented complete lysis in non-STX-producing cyanobacteria. VTD also affected cyanobacterial metabolism, and the presence of exogenous STX in the sample also ameliorated this decrease in metabolic activity, as measured by the cellular conversion of tetrazolium into formazan. Reduced primary metabolism was also recorded as a decrease in the light emissions of Vibrio fischeri exposed to VTD. Addition of STX prior to VTD resulted in a rapid and dose-dependent response to the presence of the channel blocker, with samples exhibiting resistance to the VTD effect. Our findings demonstrate that STX and VTD influence bacterial Na+ and K+ fluxes in opposite ways, and these principles can be applied to the development of a prokaryote-based STX bioassay.