120 research outputs found
Cell cycle dynamics and the physiology of saxitoxin biosynthesis in Alexandrium fundyense (Dinophyceae)
Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution March 1998The mechanism of saxitoxin (STX) biosynthesis in marine dinoflagellates of the genus
Alexandrium is still unknown. The aim of this thesis was to analyze novel aspects of
toxigenesis during the cell cycle in Alexandrium and to apply molecular techniques to gain
new insights on the genetics and regulation of STX biosynthesis.
Synchronized cultures of A. fundyense were studied to determine the dynamics of toxin
production throughout the cell cycle. Toxin production was discontinuous, was induced
by light and always occurred during a period of approximately eight to ten hours in early
G1. Analysis of the cell cycle dynamics suggests the existence of two transition points: one
at the beginning of G1, which is light-dependent and holds the cells in a Go-like period,
and a second one at the end of G1, which is size-dependent and arrests the cells in G1. A
model of the cell cycle of A. fundyense is proposed in which progression through the cell
cycle can be arrested at two different transition points located in G1 and toxin production is
induced by light during G1. The effects of temperature and phosphate limitation on the linkage between changes in
the duration of the cell cycle stages and toxicity were studied in semi-continuous cultures of
A. fundyense. A direct correlation between G1 duration and toxin content was observed,
along with a clear uncoupling of toxin accumulation from the Sand G2 phases of the cell
cycle. In both experiments, toxin production rates remained constant for the respective
range of conditions, implying that the variations in toxin content observed were a result of
increasing periods of biosynthetic activity. Phosphate limitation enhanced toxin production
rates and affected interconversions among STX derivatives in several ways: oxidations to
yield the hydroxy-series of STXs were phosphate-dependent while sulfatation reactions
were not.
Differential Display (DD) analysis was applied to the identification of genes that were
up- or downregulated during toxigenesis in synchronized cultures of A. fundyense. Three
genes were isolated: S-adenosy lhomocysteine hydrolase, methionine aminopeptidase and a
histone-like protein. None could be directly correlated to toxigenesis but instead relate to
general cellular metabolism
Biosynthesis and Molecular Genetics of Polyketides in Marine Dinoflagellates
Marine dinoflagellates are the single most important group of algae that produce toxins, which have a global impact on human activities. The toxins are chemically diverse, and include macrolides, cyclic polyethers, spirolides and purine alkaloids. Whereas there is a multitude of studies describing the pharmacology of these toxins, there is limited or no knowledge regarding the biochemistry and molecular genetics involved in their biosynthesis. Recently, however, exciting advances have been made. Expressed sequence tag sequencing studies have revealed important insights into the transcriptomes of dinoflagellates, whereas other studies have implicated polyketide synthase genes in the biosynthesis of cyclic polyether toxins, and the molecular genetic basis for the biosynthesis of paralytic shellfish toxins has been elucidated in cyanobacteria. This review summarises the recent progress that has been made regarding the unusual genomes of dinoflagellates, the biosynthesis and molecular genetics of dinoflagellate toxins. In addition, the evolution of these metabolic pathways will be discussed, and an outlook for future research and possible applications is provided
Application of COMPOCHIP Microarray to Investigate the Bacterial Communities of Different Composts
A microarray spotted with 369 different 16S rRNA gene probes specific to microorganisms involved in the degradation process of organic waste during composting was developed. The microarray was tested with pure cultures, and of the 30,258 individual probe-target hybridization reactions performed, there were only 188 false positive (0.62%) and 22 false negative signals (0.07%). Labeled target DNA was prepared by polymerase chain reaction amplification of 16S rRNA genes using a Cy5-labeled universal bacterial forward primer and a universal reverse primer. The COMPOCHIP microarray was applied to three different compost types (green compost, manure mix compost, and anaerobic digestate compost) of different maturity (2, 8, and 16 weeks), and differences in the microorganisms in the three compost types and maturity stages were observed. Multivariate analysis showed that the bacterial composition of the three composts was different at the beginning of the composting process and became more similar upon maturation. Certain probes (targeting Sphingobacterium, Actinomyces, Xylella/Xanthomonas/ Stenotrophomonas, Microbacterium, Verrucomicrobia, Planctomycetes, Low G + C and Alphaproteobacteria) were more influential in discriminating between different composts. Results from denaturing gradient gel electrophoresis supported those of microarray analysis. This study showed that the COMPOCHIP array is a suitable tool to study bacterial communities in composts
Discovery of Nuclear-Encoded Genes for the Neurotoxin Saxitoxin in Dinoflagellates
Saxitoxin is a potent neurotoxin that occurs in aquatic environments worldwide.
Ingestion of vector species can lead to paralytic shellfish poisoning, a severe
human illness that may lead to paralysis and death. In freshwaters, the toxin is
produced by prokaryotic cyanobacteria; in marine waters, it is associated with
eukaryotic dinoflagellates. However, several studies suggest that saxitoxin is
not produced by dinoflagellates themselves, but by co-cultured bacteria. Here,
we show that genes required for saxitoxin synthesis are encoded in the nuclear
genomes of dinoflagellates. We sequenced >1.2×106 mRNA
transcripts from the two saxitoxin-producing dinoflagellate strains
Alexandrium fundyense CCMP1719 and A.
minutum CCMP113 using high-throughput sequencing technology. In
addition, we used in silico transcriptome analyses, RACE, qPCR
and conventional PCR coupled with Sanger sequencing. These approaches
successfully identified genes required for saxitoxin-synthesis in the two
transcriptomes. We focused on sxtA, the unique starting gene of
saxitoxin synthesis, and show that the dinoflagellate transcripts of
sxtA have the same domain structure as the cyanobacterial
sxtA genes. But, in contrast to the bacterial homologs, the
dinoflagellate transcripts are monocistronic, have a higher GC content, occur in
multiple copies, contain typical dinoflagellate spliced-leader sequences and
eukaryotic polyA-tails. Further, we investigated 28 saxitoxin-producing and
non-producing dinoflagellate strains from six different genera for the presence
of genomic sxtA homologs. Our results show very good agreement
between the presence of sxtA and saxitoxin-synthesis, except in
three strains of A. tamarense, for which we amplified
sxtA, but did not detect the toxin. Our work opens for
possibilities to develop molecular tools to detect saxitoxin-producing
dinoflagellates in the environment
Effects of nutrients, salinity, pH and light:dark cycle on the production of reactive oxygen species in the alga Chattonella marina
Author Posting. © Elsevier B.V., 2007. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Journal of Experimental Marine Biology and Ecology 346 (2007): 76-86, doi:10.1016/j.jembe.2007.03.007.Experiments were carried out to investigate the effects of nutrients, salinity, pH and light:dark cycle on growth rate and production of reactive oxygen species (ROS) by Chattonella marina, a harmful algal bloom (HAB) species that often causes fish kills. Different nitrogen forms (organic-N and inorganic-N), N:P ratios, light:dark cycles and salinity significantly influenced algal growth, but not ROS production. However, iron concentration and pH significantly affected both growth and ROS production in C. marina. KCN (an inhibitor of mitochondrial respiration) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (an inhibitor of photosynthesis) had no significant effects on ROS production. Vitamin K3 (a plasma membrane electron shuttle) enhanced ROS production while its antagonist, dicumarol, decreased ROS production. Taken together, our results suggest that ROS production by C. marina is related to a plasma membrane enzyme system regulated by iron availability but is independent of growth, photosynthesis, availability of macronutrients, salinity and irradiance.The work described in this paper was supported by a CERG grant from the University Grants Committee of the Hong Kong Special Administrative Region, China to RSSW (Project No. 9040864). Support for DMA is provided by U.S. National Science Foundation grant # OCE-0136861
The life history of the toxic marine dinoflagellate Protoceratium reticulatum (Gonyaulacales) in culture
Asexual and sexual life cycle events were studied in cultures of the toxic marine dinoflagellate Protoceratium reticulatum. Asexual division by desmoschisis was characterized morphologically and changes in DNA content were analyzed by flow cytometry. The results indicated that haploid cells with a C DNA content occurred only during the light period whereas a shift from a C to a 2C DNA content (indicative of S phase) took place only during darkness. The sexual life cycle was documented by examining the mating type as well as the morphology of the sexual stages and nuclei. Gamete fusion resulted in a planozygote with two longitudinal flagella, but longitudinally biflagellated cells arising from planozygote division were also observed, so one of the daughter cells retained two longitudinal flagella while the other daughter cell lacked them. Presumed planozygotes (identified by their longitudinally biflagellated form) followed two life-cycle routes: division and encystment (resting cyst formation). Both the division of longitudinally biflagellated cells and resting cyst formation are morphologically described herein. Resting cyst formation through sexual reproduction was observed in 6.1% of crosses and followed a complex heterothallic pattern. Clonal strains underwent sexuality (homothallism for planozygote formation and division) but without the production of resting cysts. Ornamental processes of resting cysts formed from the cyst wall under an outer balloon-shaped membrane and were fully developed in <1 h. Obligatory dormancy period was of ∼4 months. Excystment resulted in a large, rounded, pigmented, longitudinally biflagellated but motionless, thecate germling that divided by desmoschisis. Like the planozygote, the first division of the germling yielded one longitudinally biflagellated daughter cell and another without longitudinal flagella. The longitudinal biflagellation state of both sexual stages and of the first division products of these cells is discussed.Postprin
Origin of Saxitoxin Biosynthetic Genes in Cyanobacteria
BACKGROUND:Paralytic shellfish poisoning (PSP) is a potentially fatal syndrome associated with the consumption of shellfish that have accumulated saxitoxin (STX). STX is produced by microscopic marine dinoflagellate algae. Little is known about the origin and spread of saxitoxin genes in these under-studied eukaryotes. Fortuitously, some freshwater cyanobacteria also produce STX, providing an ideal model for studying its biosynthesis. Here we focus on saxitoxin-producing cyanobacteria and their non-toxic sisters to elucidate the origin of genes involved in the putative STX biosynthetic pathway. METHODOLOGY/PRINCIPAL FINDINGS:We generated a draft genome assembly of the saxitoxin-producing (STX+) cyanobacterium Anabaena circinalis ACBU02 and searched for 26 candidate saxitoxin-genes (named sxtA to sxtZ) that were recently identified in the toxic strain Cylindrospermopsis raciborskii T3. We also generated a draft assembly of the non-toxic (STX-) sister Anabaena circinalis ACFR02 to aid the identification of saxitoxin-specific genes. Comparative phylogenomic analyses revealed that nine putative STX genes were horizontally transferred from non-cyanobacterial sources, whereas one key gene (sxtA) originated in STX+ cyanobacteria via two independent horizontal transfers followed by fusion. In total, of the 26 candidate saxitoxin-genes, 13 are of cyanobacterial provenance and are monophyletic among the STX+ taxa, four are shared amongst STX+ and STX-cyanobacteria, and the remaining nine genes are specific to STX+ cyanobacteria. CONCLUSIONS/SIGNIFICANCE:Our results provide evidence that the assembly of STX genes in ACBU02 involved multiple HGT events from different sources followed presumably by coordination of the expression of foreign and native genes in the common ancestor of STX+ cyanobacteria. The ability to produce saxitoxin was subsequently lost multiple independent times resulting in a nested relationship of STX+ and STX- strains among Anabaena circinalis strains
Multi-level analysis of the gut-brain axis shows autism spectrum disorder-associated molecular and microbial profiles
Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by heterogeneous cognitive, behavioral and communication impairments. Disruption of the gut-brain axis (GBA) has been implicated in ASD although with limited reproducibility across studies. In this study, we developed a Bayesian differential ranking algorithm to identify ASD-associated molecular and taxa profiles across 10 cross-sectional microbiome datasets and 15 other datasets, including dietary patterns, metabolomics, cytokine profiles and human brain gene expression profiles. We found a functional architecture along the GBA that correlates with heterogeneity of ASD phenotypes, and it is characterized by ASD-associated amino acid, carbohydrate and lipid profiles predominantly encoded by microbial species in the genera Prevotella, Bifidobacterium, Desulfovibrio and Bacteroides and correlates with brain gene expression changes, restrictive dietary patterns and pro-inflammatory cytokine profiles. The functional architecture revealed in age-matched and sex-matched cohorts is not present in sibling-matched cohorts. We also show a strong association between temporal changes in microbiome composition and ASD phenotypes. In summary, we propose a framework to leverage multi-omic datasets from well-defined cohorts and investigate how the GBA influences ASD
Enhancing photosynthetic production of ethylene in genetically engineered Synechocystis sp. PCC 6803
The Hidden Sexuality of Alexandrium Minutum: An Example of Overlooked Sex in Dinoflagellates
Dinoflagellates are haploid eukaryotic microalgae in which rapid proliferation causes dense
blooms, with harmful health and economic effects to humans. The proliferation mode is
mainly asexual, as the sexual cycle is believed to be rare and restricted to stressful environmental
conditions. However, sexuality is key to explaining the recurrence of many dinoflagellate
blooms because in many species the fate of the planktonic zygotes (planozygotes)
is the formation of resistant cysts in the seabed (encystment). Nevertheless, recent
research has shown that individually isolated planozygotes in the lab can enter other routes
besides encystment, a behavior of which the relevance has not been explored at the population
level. In this study, using imaging flow cytometry, cell sorting, and Fluorescence In
Situ Hybridization (FISH), we followed DNA content and nuclear changes in a population of
the toxic dinoflagellate Alexandrium minutum that was induced to encystment. Our results
first show that planozygotes behave like a population with an “encystment-independent”
division cycle, which is light-controlled and follows the same Light:Dark (L:D) pattern as the
cycle governing the haploid mitosis. Resting cyst formation was the fate of just a small fraction
of the planozygotes formed and was restricted to a period of strongly limited nutrient
conditions. The diploid-haploid turnover between L:D cycles was consistent with two-step
meiosis. However, the diel and morphological division pattern of the planozygote division
also suggests mitosis, which would imply that this species is not haplontic, as previously
considered, but biphasic, because individuals could undergo mitotic divisions in both the
sexual (diploid) and the asexual (haploid) phases. We also report incomplete genome duplication
processes. Our work calls for a reconsideration of the dogma of rare sex in
dinoflagellates.Versión del edito
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