Co-occurrence of microcystin and microginin congeners in Brazilian strains of Microcystis sp.

Species of Microcystis are the most common bloom-forming cyanobacteria in several countries. Despite extensive studies regarding the production of bioactive cyanopeptides in this genus, there are limited data on isolated strains from Brazil. Three Microcystis sp. strains were isolated from the Salto Grande Reservoir (LTPNA01, 08 and 09) and investigated for the presence of mcy genes, microcystins and other cyanopeptides. Microcystin and microginin production was confirmed in two isolates using high-resolution tandem mass spectrometry after electrospray ionization (ESI-Q-TOF), and the structures of two new microginin congeners were proposed (MG756 Ahda-Val-Leu-Hty-Tyr and MG770 MeAhda-Val-Leu-Hty-Tyr). The biosynthesis profile of the identified cyanopeptides was evaluated at different growth phases via a newly developed HPLC-UV method. Results demonstrated no substantial differences in the production of microcystins and microginins after data normalization to cell quota, suggesting a constitutive biosynthesis. This study represents the first confirmed co-production of microginins and microcystins in Brazilian strains of Microcystis sp. and highlights the potential of Brazilian cyanobacteria as a source of natural compounds with pharmaceutical interest.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo) [2010/15651-9, 2010/15696-2]CAPES (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)CONICYT (Programa de Cooperacion Cientifica Internacional)CONICYT (Programa de Cooperacion Cientifica Internacional)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico

Metagenome sequencing of the microbial community of a solar saltern crystallizer pond at cáhuil lagoon, chile.

Cáhuil Lagoon in central Chile harbors distinct microbial communities in various solar salterns that are arranged as interconnected ponds with increasing salt concentrations. Here, we report the metagenome of the 3.0- to 0.2-µm fraction of the microbial community present in a crystallizer pond with 34% salinity

Distinctive Archaeal Composition of an Artisanal Crystallizer Pond and Functional Insights Into Salt-Saturated Hypersaline Environment Adaptation

Hypersaline environments represent some of the most challenging settings for life on Earth. Extremely halophilic microorganisms have been selected to colonize and thrive in these extreme environments by virtue of a broad spectrum of adaptations to counter high salinity and osmotic stress. Although there is substantial data on microbial taxonomic diversity in these challenging ecosystems and their primary osmoadaptation mechanisms, less is known about how hypersaline environments shape the genomes of microbial inhabitants at the functional level. In this study, we analyzed the microbial communities in five ponds along the discontinuous salinity gradient from brackish to salt-saturated environments and sequenced the metagenome of the salt (halite) precipitation pond in the artisanal Cáhuil Solar Saltern system. We combined field measurements with spectrophotometric pigment analysis and flow cytometry to characterize the microbial ecology of the pond ecosystems, including primary producers and applied metagenomic sequencing for analysis of archaeal and bacterial taxonomic diversity of the salt crystallizer harvest pond. Comparative metagenomic analysis of the Cáhuil salt crystallizer pond against microbial communities from other salt-saturated aquatic environments revealed a dominance of the archaeal genus Halorubrum and showed an unexpectedly low abundance of Haloquadratum in the Cáhuil system. Functional comparison of 26 hypersaline microbial metagenomes revealed a high proportion of sequences associated with nucleotide excision repair, helicases, replication and restriction-methylation systems in all of them. Moreover, we found distinctive functional signatures between the microbial communities from salt-saturated (>30% [w/v] total salinity) compared to sub-saturated hypersaline environments mainly due to a higher representation of sequences related to replication, recombination and DNA repair in the former. The current study expands our understanding of the diversity and distribution of halophilic microbial populations inhabiting salt-saturated habitats and the functional attributes that sustain them

Hemolymph Bacterial Community of Pacific Oyster (Crassostrea gigas) in Response to Long-Term Hypercapnia

In this paper I will study the changes in the bacterial community associated to C. gigas in response to the physiological changes triggered by simulated future pCO2 conditions. Specifically changes of the community associated to the hemolymph will be analyzed in early adults stages of C.gigas and its will be correlated with the physiological changes of the oyster under induced long-term hypercapni

Immunofluorescence-LM of proteins in intact trichomes.

<p>Cultures grown in MLA₀ and a 12L/12D cycle were sampled 3 h after the beginning of the light phase. Immunofluorescence was performed on <i>C. raciborskii</i> using antibodies against (A) NtcA, (B) FtsZ, (C) RbcL proteins and against the NifH protein in both (D) <i>C. raciborskii</i> and (E) <i>Anabaena</i> PCC7120. (F) <i>A. variabilis</i> ATCC29413 grown in BG11₀ and a 12L/12D cycle was used as a positive control for NifH (<i>nif</i>2 nitrogenase) presence in vegetative cells (when sampled 4 h in the dark phase). Arrows indicate the positions of heterocysts.</p

Phylogenetic analysis of PatAL and HetF homologs in <i>C. raciborskii</i>.

<p>Maximum-likelihood phylogenetic trees of (A) PatA, (B) PatL, and (C) HetF. Branches are colored by their support value (Shimodaira-Hasegawa test, low [blue] to high [red]). <i>C. raciborskii</i> and <i>R. brookii</i> sequences are colored in pink for clarity. Clades including the canonical heterocyst differentiation proteins are shaded in green while non-canonical heterocystous clades are shaded in yellow. Scale bar shows expected substitutions per site. In (C), the “[01]” suffix for Rapb|28289569 indicates that the sequence is a concatenation of two neighboring ORFs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051682#pone.0051682.s002" target="_blank">Information S1</a> for details).</p

The cyanobacterium <i>C. raciborskii</i> grown under diazotrophic conditions.

<p>(A) Light micrograph of <i>C. raciborskii</i> trichomes, magnified 40x. Note the terminal heterocysts at the end of the trichomes (white arrows); one heterocyst is magnified. (B) Percentage of trichomes with 10 to >100 vegetative cells are grouped according to their trichome length (no. of vegetative cells per trichome). The bars give the average numbers in 400 trichomes from 4 independent cultures. Standard deviation is given. The dashed line denotes the point where all trichomes have developed two end heterocysts. (C) The growth of <i>C. raciborskii</i> cultures, grown with 2 mM of NO₃⁻ to an OD₇₅₀ of 0.2, washed and transferred to new medium under the denoted light regime. Each point corresponds to the mean of 4 biological replicates. Standard deviation is given. (D) Western-blot analysis of NifH expression during a 12L/12D cycle (above). The NifH western-blot membrane was stripped and re-labelled with an antibody against the 30S ribosomal protein S1, revealing protein levels loaded in each well (below). The closest molecular marker band is indicated, and black and white bars represent the light and dark phase of the cycle. (E) Nitrogenase activity was analyzed by ARA for <i>C. raciborskii</i> under diazotrophic growth and continuous light (MLA₀ Cont light), diazotrophic growth and 12L/12D cycle (MLA₀ 12/12), and grown in MLA_N under 12L/12D cycle (MLA_N 12/12). Oxygen concentration was measured for MLA₀ 12/12 cultures and normalized to protein content. Each point corresponds to the mean of three biological replicates where standard deviation is shown.</p

Cell enrichment purity assessment and nitrogenase expression in heterocyst and vegetative cells of <i>C. raciborskii</i>.

<p>Heterocysts and vegetative cells of <i>C. raciborskii</i> were separated, proteins were extracted and cDNA was generated for both cell fractions. (A) RT-PCR amplification for <i>dev</i>A (lanes 1–4) and <i>hgl</i>D (lanes 5–8). (B) PCR amplification of <i>rbc</i>S (lanes 1–4) and <i>fts</i>Z (lanes 5–8). (C) RbcL levels. (D) FtsZ (upper) and S1 protein levels (lower). (E) RT-PCR amplification of <i>nif</i>H (lanes 1–4) and a fragment of the 16S rRNA (lanes 5–8). (F) NifH levels (upper) and S1 protein levels (lower). Western-blot membranes were stripped and re-labelled with the 30S ribosomal protein S1 to verify loading similarities. Protein levels and selected gene expression levels were determined in enriched heterocysts (Het/H) or vegetative cell (Veg/V) fractions. Protein extracts from non-fractionated trichomes of <i>C. raciborskii</i> were used as a positive control (+). The closest molecular marker band is indicated. <i>C. raciborskii</i> DNA, and the RNA used to generate the cDNA, were used as positive (+), and negative (−) controls respectively. M1 and M2 denote DNA size markers (bp).</p

Dinitrogen Fixation Is Restricted to the Terminal Heterocysts in the Invasive Cyanobacterium <em>Cylindrospermopsis raciborskii</em> CS-505

<div><p>The toxin producing nitrogen-fixing heterocystous freshwater cyanobacterium <em>Cylindrospermopsis raciborskii</em> recently radiated from its endemic tropical environment into sub-tropical and temperate regions, a radiation likely to be favored by its ability to fix dinitrogen (diazotrophy). Although most heterocystous cyanobacteria differentiate regularly spaced intercalary heterocysts along their trichomes when combined nitrogen sources are depleted, <em>C. raciborskii</em> differentiates only two terminal heterocysts (one at each trichome end) that can reach &gt;100 vegetative cells each. Here we investigated whether these terminal heterocysts are the exclusive sites for dinitrogen fixation in <em>C. raciborskii</em>. The highest nitrogenase activity and NifH biosynthesis (western-blot) were restricted to the light phase of a 12/12 light/dark cycle. Separation of heterocysts and vegetative cells (sonication and two-phase aqueous polymer partitioning) demonstrated that the terminal heterocysts are the sole sites for <em>nifH</em> expression (RT-PCR) and NifH biosynthesis. The latter finding was verified by the exclusive localization of nitrogenase in the terminal heterocysts of intact trichomes (immunogold-transmission electron microscopy and <em>in situ</em> immunofluorescence-light microscopy). These results suggest that the terminal heterocysts provide the combined nitrogen required by the often long trichomes (&gt;100 vegetative cells). Our data also suggests that the terminal-heterocyst phenotype in <em>C. raciborskii</em> may be explained by the lack of a <em>patL</em> ortholog. These data help identify mechanisms by which <em>C. raciborskii</em> and other terminal heterocyst-forming cyanobacteria successfully inhabit environments depleted in combined nitrogen.</p> </div