42 research outputs found
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
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
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
Relationship of proteomic variation and toxin synthesis in the dinoflagellate Alexandrium tamarense CI01 under phosphorus and inorganic nitrogen limitation
Alexandrium fundyense cyst viability and germling survival in light vs. dark at a constant low temperature
Broad anti-pathogen potential of DEAD box RNA helicase eIF4A-targeting rocaglates
Abstract Inhibition of eukaryotic initiation factor 4A has been proposed as a strategy to fight pathogens. Rocaglates exhibit the highest specificities among eIF4A inhibitors, but their anti-pathogenic potential has not been comprehensively assessed across eukaryotes. In silico analysis of the substitution patterns of six eIF4A1 aa residues critical to rocaglate binding, uncovered 35 variants. Molecular docking of eIF4A:RNA:rocaglate complexes, and in vitro thermal shift assays with select recombinantly expressed eIF4A variants, revealed that sensitivity correlated with low inferred binding energies and high melting temperature shifts. In vitro testing with silvestrol validated predicted resistance in Caenorhabditis elegans and Leishmania amazonensis and predicted sensitivity in Aedes sp., Schistosoma mansoni, Trypanosoma brucei, Plasmodium falciparum, and Toxoplasma gondii. Our analysis further revealed the possibility of targeting important insect, plant, animal, and human pathogens with rocaglates. Finally, our findings might help design novel synthetic rocaglate derivatives or alternative eIF4A inhibitors to fight pathogens