A novel gene encoding amidinotransferase in the cylindrospermopsin producing cyanobacterium Aphanizomenon ovalisporum

The hepatotoxin cylindrospermopsin is produced by several cyanobacteria species, which may flourish in tropical and sub-tropical lakes. Biosynthesis of cylindrospermopsin is poorly understood but its chemical nature, and feeding experiments with stable isotopes, suggested that guanidinoacetic acid is the starter unit and indicated involvement of a polyketide synthase. We have identified a gene encoding an amidinotransferase from the cylindrospermopsin producing cyanobacterium Aphanizomenon ovalisporum. This is the first report on an amidinotransferase gene in cyanobacteria. It is likely to be involved in the formation of guanidinoacetic acid. The aoaA is located in a genomic region bearing genes encoding a polyketide synthase and a peptide synthetase, further supporting its putative role in cylindrospermopsin biosynthesis.     

Capillary electrophoretic assay and purification of cylindrospermopsin, a cyanobacterial toxin from Aphanizomenon ovalisporum, by plant test (blue-green Sinapis test)

Toxic cyanobacteria are known to produce cyanotoxins, toxic secondary metabolites. In recent years the cylindrospermopsin (tricyclic guanidinyl hydroxymethyluracil)-producing organisms Aphanizomenon ovalisporum, Cylindrospermopsis raciborskii, and Umezakia natans have been inhabiting polluted fresh waters. Cylindrospermopsin, a potent hepatotoxic cyanotoxin, has been implicated in cases of human poisoning as well. This study describes the isolation and purification of cylindrospermopsin from A. ovalisporum with the help of a slightly modified Blue-Green Sinapis Test, a plant test suitable for determining the cyanotoxin content of chromatographic fractions besides plankton samples. The recent modification, using microtiter plates for the assay, improves the method and reduces the amount of sample needed for the assay. This approach proved that plant growth and metabolism, at least in the case of etiolated Sinapis alba seedlings, are inhibited by cylindrospermopsin. The establishment of capillary electrophoresis of cylindrospermopsin and consideration of the results reported here lead us to the expectation that capillary electrophoresis of cylindrospermopsin may be a powerful and useful analytical method for investigating cyanobacterial blooms for potential cylindrospermopsin content and toxicity. Confirmation of chemical identity of the purified compound is performed by UV spectrophotometry, NMR, and MALDI-TOF.     

Alteration of cylindrospermopsin production in sulfate- or phosphate-starved cyanobacterium Aphanizomenon ovalisporum

The effect of sulfate and phosphate deprivation on cell growth and cylindrospermopsin level was studied in Aphanizomenon ovalisporum ILC-164. Sulfate starvation induced a characteristic reduction of cylindrospermopsin pool size on the basis of cell number and unit of dry mass of culture. Phosphorous starvation of A. ovalisporum cultures induced a lesser reduction of cylindrospermopsin pool size. This divergence in the pool size of cylindrospermopsin may be the consequence of different growth rate. To show the metabolic changes concomitant with reduction of cylindrospermopsin pool size were obtained by measurement of ATP sulfurylase and alkaline phosphatase activity. The present study is the first concerning the cylindrospermopsin content under sulfate starvation and discusses it in relation to phosphorous starvation.     

Diversity of and selection acting on cylindrospermopsin cyrB gene adenylation domain sequences in Florida

Aphanizomenon ovalisporum is the only confirmed cylindrospermopsin producer identified in the United States to date. On the other hand, Cylindrospermopsis raciborskii is a prominent feature of many lakes in Florida and other regions of the United States. To see the variation in cylindrospermopsin cyrB gene adenylation domain sequences and possibly discover new cylindrospermopsin producers, we collected water samples for a 3-year period from 17 different systems in Florida. Positive amplicons were cloned and sequenced, revealing that approximately 92% of sequences were A. ovalisporum-like (>99% identity). Interestingly, 6% of sequences were very similar (>99% identity) to cyrB sequences of C. raciborskii from Australia and of Aphanizomenon sp. from Germany. Neutrality tests suggest that A. ovalisporum-like cyrB adenylation domain sequences are under purifying selection, with abundant low-frequency polymorphisms within the population. On the other hand, when compared between species by codon-based methods, amino acids of CyrB also seem to be under purifying selection, in accordance with the one proposed amino acid thought to be activated by the CyrB adenylation domain.     

Cyanotoxins: a poison that frees phosphate

Autotrophic organisms obtain phosphorus from the environment by secreting alkaline phosphatases that act on esters, resulting in inorganic phosphate that is then taken up. New work shows that the cyanobacterium Aphanizomenon ovalisporum obtains inorganic phosphate by secreting the cyanotoxin cylindrospermopsin, which induces alkaline phosphatase in other phytoplankton species.     

IDENTIFICATION OF CYLINDROSPERMOPSIN IN APHANIZOMENON OVALISPORUM (CYANOPHYCEAE) ISOLATED FROM LAKE KINNERET,ISRAEL1

Aphanizomenon ovalisporum (Forti) was identified and isolated from Lake Kinneret upon its first appearance as a dominant bloom in late 1994. This cyanobacterial species, not previously known to be toxic, was evaluated by a commonly used mouse bioassay and was demonstrated to induce toxic symptoms that were distinguishable from the typical symptoms of the neurotoxins previously reported in Aphanizomenon flos-aquae (L.) Ralfs. Mice died 5–24 h after crude extracts were injected intraperitoneally, and the LD₅₀ value was estimated as 465 mg dry wt biomass · kg^?1 mouse. A toxicity-guided fractionation of the active extract indicated that the potent substance is polar an nature. The structure of the active compound was determined by its mass spectrometry and NMR data. The compound was found to be the sulfate-guanidinium zwitterion, cylindrospermopsin, previously isolated from the cyanobacterium Cylindrospermopsis raciborskii (Woloszynska) and recently also reported in Umezakia natans (Watanabe). This is the first time that Aphanizomenon ovalisporum has been reported to contain a toxic compound.     

Consumption of cyanobacteria by roach (Rutilus rutilus): useful or harmful to the fish?

1. The ability of roach to use cyanobacterial food is generally believed to be one reason for the dominance of roach over perch in eutrophic European lakes. The aim of this study was to test whether cyanobacteria really are a suitable food for juvenile roach. Special attention was paid to differences between the two cyanobacteria species Aphanizomenon and Microcystis which are common in eutrophic lakes and are ingested by roach there.
2. We performed growth and behaviour experiments with juvenile roach fed with zooplankton and the different cyanobacteria. Growth rate with Aphanizomenon was lower than with Daphnia but significantly higher than without food, whereas growth rate with Microcystis was as low as without food.
3. In cultivation experiments of roach faeces, Microcystis was found not to have been digested and grew exponentially after passing through the gut whereas Aphanizomenon stayed at low biomass. Differences in growth were not related to the toxin content of cyanobacteria. Investigations of roach motility showed no differences whether fed with Aphanizomenon or Microcystis .
4. In contrast to Microcystis , Aphanizomenon can be regarded as a suitable food source for juvenile roach probably because of its better digestability. We conclude that the ability to feed on cyanobacteria is not a general competitive advantage for roach, but the outcome depends on the species composition of the cyanobacteria.     

Molecular characterization of the transition state regulator AbrB from Bacillus stearothermophilus

The Bacillus subtilis transition state regulator AbrB(su) is a DNA-binding protein that acts on several genes either as activator, repressor, or preventer. However, among genes under its control, neither common binding sites could be identified nor could the structural features of this broad and specific interaction be elucidated. Attempts to elucidate these interesting features by crystallizing AbrB(su) have failed so far. Therefore, to solve this problem, we focused in this work on identifying an AbrB(su) homologue from Bacillus stearothermophilus. Using a novel method, the entire abrB(st) gene of B. stearothermophilus was cloned and sequenced. The gene encodes a 95 amino acid protein that shows 77% identity and 85% similarity to the mesophilic B. subtilis protein. A calmodulin binding peptide-tagged fusion of the thermophilic gene was constructed for overexpression and efficient affinity column purification of the AbrB(st) protein. The purified protein showed, after removal of the tag, an oligomerization behavior through hexamer formation that is essential for its DNA binding activity.     

Functional Modeling and Phylogenetic Distribution of Putative Cylindrospermopsin Biosynthesis Enzymes

The alkaloid cylindrospermopsin is the most recently discovered cyanotoxin and has caused epidemic outbreaks of human poisoning. Cylindrospermopsin producing cyanobacteria have in recent times appeared in countries all over the world where they had not been observed previously and, thus, represent a global public health concern. Three putative cylindrospermopsin biosynthesis genes, encoding an amidinotransferase (aoaA), a nonribosomal peptide synthetase (aoaB), and a polyketide synthase (aoaC), have been described. Most cyanotoxins are the product of nonribosomal peptide and polyketide synthesis, but the involvement of an amidinotransferase is novel. In the present study, functional modeling was carried out to gain insight into the mechanism of precursor recruitment in cylindrospermopsin biosynthesis. In addition, the molecular phylogenies of putative cylindrospermopsin biosynthesis genes and producer organisms were determined. The model indicated that AoaA may catalyze the formation of guanidino acetate from glycine and arginine. The catalytic site of the AoaB adenylation domain provided two aspartate residues, instead of the usual one, which may be involved in the binding of the guanidino moiety of guanidino acetate. Molecular phylogenetic analysis grouped cylindrospermopsin producing cyanobacteria into two divergent groups. Although the phylogeny of the cylindrospermopsin biosynthesis genes followed that of the producer organisms, they were less divergent, which may indicate the recent horizontal transfer of these genes. [Reviewing Editor: Dr. Martin Kreitman]     

Study of nitrogen fixation in microbial communities of oil-contaminated marine sediment microcosms

Aerobic microbial degradation of pollutant oil (petroleum) in aquatic environments is often severely limited by the availability of combined nitrogen. We therefore studied whether the microbial community enriched in marine sediment microcosms with an added oil layer and exposure to light harboured nitrogenase activity. The acetylene reduction (AR) assay indeed indicated active nitrogenase; however, similar activity was observed in oil-free control microcosms. In both microcosms, the AR rate was significantly reduced upon a dark shift, indicating that enriched cyanobacteria were the dominant diazotrophs. Analysis of structural dinitrogenase reductase genes (nifH) amplified from both microcosms indeed revealed NifH sequences related mostly to those of heterocystous cyanobacteria. NifH sequences typically affiliating with those of heterotrophic bacteria were more frequently retrieved from the oil-containing sediment. Expression analyses showed that mainly nifH genes similar to those of heterocystous cyanobacteria were expressed in the light. Upon a dark shift, nifH genes related to those of non-heterocystous cyanobacteria were expressed. Expression of nifH assignable to heterotrophs was apparently not significant. It is concluded that cyanobacteria are the main contributors of fixed nitrogen to oil-contaminated and pristine sediments if nitrogen is a limiting factor and if light is available. Hence, also the oil-degrading heterotrophic community may thus receive a significant part of combined nitrogen from cyanobacteria, even though oil vice versa apparently does not stimulate an additional nitrogen fixation in the enriched community.     

Detection of a homotetrameric structure and protein–protein interactions of Paracoccidioides brasiliensis formamidase lead to new functional insights

Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic mycosis in Latin America. Formamidases hydrolyze formamide, putatively plays a role in fungal nitrogen metabolism. An abundant 45-kDa protein was identified as the P. brasiliensis formamidase. In this study, recombinant formamidase was overexpressed in bacteria and a polyclonal antibody to this protein was produced. We identified a 180-kDa protein species reactive to the antibody produced in mice against the P. brasiliensis recombinant purified formamidase of 45 kDa. The 180-kDa purified protein yielded a heat-denatured species of 45 kDa. Both protein species of 180 and 45 kDa were identified as formamidase by peptide mass fingerprinting using MS. The identical mass spectra generated by the 180 and the 45-kDa protein species indicated that the fungal formamidase is most likely homotetrameric in its native conformation. Furthermore, the purified formamidase migrated as a protein of 191 kDa in native polyacrylamide gel electrophoresis, thus revealing that the enzyme forms a homotetrameric structure in its native state. This enzyme is present in the fungus cytoplasm and the cell wall. Use of a yeast two-hybrid system revealed cell wall membrane proteins, in addition to cytosolic proteins interacting with formamidase. These data provide new insights into formamidase structure as well as potential roles for formamidase and its interaction partners in nitrogen metabolism.     

Aphanizomenon ovalisporum (Forti) in Lake Kinneret, Israel

The filamentous cyanobacterium Aphanizomenon ovalisporum wasobserved for the first time in Lake Kinneret in August 1994and formed a prominent bloom from September through October.Aphanizomenon ovalisporum reappeared in diminished amounts inthe summer and fall of 1995. These events are the first recordof significant quantities of a potentially toxic nitrogen-fixingcyanobacterium in this lake. No definite provenance of inoculumhas been identified, although A.ovalisporum was also observedin a newly reflooded area (Lake Agmon) in the catchment. Unusuallyhigh water temperatures and low wind inputs were observed priorto and during the A.ovalisporum bloom period. These, togetherwith possibly enhanced availability of phosphorus or other growthfactors, may have contributed to the cyanobacterium growth in1994. Phosphorus limi tation, as indicated by high cellularalkaline phosphatase activity, the onset of stormy conditionsand a fall in water temperatures led to the demise of the 1994bloom. Although the A. ovalisporum bloom in 1994 had no seriousdirect impact on water quality, the continued presence of apotentially toxic cyanobacterium in Lake Kinneret, a major nationalwater supply source, is a cause for serious concern.     

Cylindrospermopsin is not degraded by co-occurring natural bacterial communities during a 40-day study

Biodegradation of cylindrospermopsin produced by Aphanizomenon ovalisporum UAM 290 was studied. In the 40-day degradation experiment conducted, bacterial communities from two waterbodies with and without previous exposure to the toxin were used. Further, and in order to study the potential effect of other organic substrates on the degradation of cylindrospermopsin, three different sources of cylindrospermopsin were used: toxic extracts obtained by methanolic extraction and by ultrasonication in water with 5% formic acid and 0.9% NaCl and toxin naturally present in the spent media of an Aphanizomenon ovalisporum culture. Despite active growth of the bacterial population and consumption of DOC in presence of the toxic extracts, no degradation of cylindrospermopsin could be observed during the 40-day period. Considering that cylindrospermopsin is abundant in the extracellular fraction and that photodegradation in the field seems to be limited, a lack of efficient biodegradation as observed in our study could be of greatest importance and further explain the accumulation of this toxin in the dissolved fraction of the waterbodies investigated.