Horňák K., Kasalický V., Šimek K., Grossart HP. (2017): Strain-specific consumption and transformation of alga-derived dissolved organic matter by members of the Limnohabitans-C and Polynucleobacter-B clusters of Betaproteobacteria. Environ. Microbiol. 19: 4519–4535. doi:10.1111/1462-2920.13900.
We investigated changes in quality and quantity of extracellular and biomass-derived organic matter (OM) from three axenic algae (genera Rhodomonas, Chlamydomonas, Coelastrum) during growth of Limnohabitans parvus, Limnohabitans planktonicus and Polynucleobacter acidiphobus representing important clusters of freshwater planktonic Betaproteobacteria. Total extracellular and biomass-derived OM concentrations from each alga were approximately 20 mg/l and 1 mg/l respectively, from which up to 9% could be identified as free carbohydrates, polyamines, or free and combined amino acids. Carbohydrates represented 54%–61% of identified compounds of the extracellular OM from each alga. In biomass-derived OM of Rhodomonas and Chlamydomonas 71%–77% were amino acids and polyamines, while in that of Coelastrum 85% were carbohydrates. All bacteria grew on alga-derived OM of Coelastrum, whereas only Limnohabitans strains grew on OM from Rhodomonas and Chlamydomonas. Bacteria consumed 24%–76% and 38%–82% of all identified extracellular and biomass-derived OM compounds respectively, and their consumption was proportional to the concentration of each OM compound in the different treatments. The bacterial biomass yield was higher than the total identifiable OM consumption indicating that bacteria also utilized other unidentified alga-derived OM compounds. Bacteria, however, also produced specific OM compounds suggesting enzymatic polymer degradation or de novo exudation.
Voráčová K., Hájek J., Mareš J., Urajová P., Kuzma M., Cheel J., Villunger A., Kapuscik A., Bally M., Novák P., Kabeláč P., Krumschnabel G., Lukeš M., Voloshko L., Kopecký J. & Hrouzek P. (2017): The cyanobacterial metabolite nocuolin A is a natural oxadiazine that triggers apoptosis in human cancer cells. - PLoS ONE 12(3): e0172850. doi:10.1371/journal.pone.
Oxadiazines are heterocyclic compounds containing N-N-O or N-N-C-O system within a six membered ring. These structures have been up to now exclusively prepared via organic synthesis. Here, we report the discovery of a natural oxadiazine nocuolin A (NoA) that has a unique structure based on 1,2,3-oxadiazine. We have identified this compound in three independent cyanobacterial strains of genera Nostoc, Nodularia, and Anabaena and recognized the putative gene clusters for NoA biosynthesis in their genomes. Its structure was characterized using a combination of NMR, HRMS and FTIR methods. The compound was first isolated as a positive hit during screening for apoptotic inducers in crude cyanobacterial extracts. We demonstrated that NoA-induced cell death has attributes of caspase-dependent apoptosis. Moreover, NoA exhibits a potent anti-proliferative activity (0.7±4.5 µM) against several human cancer lines, with p53-mutated cell lines being even more sensitive. Since cancers bearing p53 mutations are resistant to several conventional anti-cancer drugs, NoA may offer a new scaffold for the development of drugs that have the potential to target tumor cells independent of their p53 status. As no analogous type of compound was previously described in the nature, NoA establishes a novel class of bioactive secondary metabolites.
Bednařík A, Blaser M, Matoušů A, Hekera P, and Rulík M. 2017: Effect of weir impoundments on methane dynamics in a river. Science of the Total Environment 584–585: 164–174.
We measured CH4 concentration, CH4 oxidation in the water column and total CH4 emissions to the atmosphere (diffusion and ebullition) in three weir impoundments and river reaches between them, in order to understand their role in river methane (CH4) dynamics. Sediment samples were also collected to determine CH4 consumption and production potentials together with the contribution of individual methanogenic pathways. The CH4 surface water concentration increased 7.5 times in the 16 km long river stretch. Microbial CH4 oxidation in the water column reached values ranging from 51 to 403 nmol l−1 d−1 and substantially contributed to the CH4 removal from surface water, together with CH4 emissions. The total CH4 emissions to the atmosphere varied between 0.8 and 207.1 mmol CH4 m−2 d−1 with the highest values observed upstream of the weirs (mean 68.5 ± 29.9 mmol CH4 m−2 d−1). Most of the CH4 was transported through the air-water interface by ebullition upstream of the weirs, while the ebullition accounted for 95.8 ± 2.0% of the total CH4 emissions. Both CH4 production and oxidation potential of sediments were higher upstream of the weirs compared to downstream of the weirs. The contribution of hydrogenotrophic methanogenesis to total CH4 sediment production was 36.7–89.4% and prevailed upstream of the weirs. Our findings indicate that weirs might influence river CH4 dynamics, especially by increased CH4 production and consumption by sediments, followed by increasing CH4 emissions to the atmosphere.
Salcher MM, Šimek K. 2016: Isolation and cultivation of planktonic freshwater microbes is essential for a comprehensive understanding of their ecology. Aquatic Microbial Ecology 77: 183–196. doi: 10.3354/ame01796
Representative model organisms form the basis on which biology is constructed, and pure cultures offer many opportunities for discovery. However, our view of the importance of axenic cultures changed dramatically at the turn of the last century upon realizing that the majority of environmentally relevant microbes still remains uncultured. The sequencing revolution has led us to a point where we can identify the microbial world in which we live, but many questions remain regarding the autecology of planktonic microbes and their interactions with their environment. Thus, it is essential to isolate and cultivate the key microbial players to gain a deeper insight into their ecology. If the past is a guide, the way forward in confronting the so-called ‘great plate count anomaly’ is the use of more subtle and refined approaches to culturing, using a number of methods and processes that are now becoming available. The vast amount of information accumulated from genome sequencing alone has yet to result in the isolation of the most important and abundant microbes of aquatic systems. We highlight the merits of pure cultures and discuss the critical need to integrate information from a variety of different sources to isolate planktonic microbes. We also describe how to culture bacteria of interest with a full cycle isolation approach based on targeted enrichment and illustrate the benefits of pure cultures with 2 examples of isolated representatives of freshwater Betaproteobacteria.
Pitsch, G., Adamec, L., Dirren, S., Nitsche, F., Šimek, K., Sirová, D., Posch T. The green Tetrahymena utriculariae n. sp. (Ciliophora, Oligohymenophorea) with its endosymbiotic algae (Micractinium sp.), living in the feeding traps of a carnivorous aquatic plant. J. Eukaryot. Microbiol. (Accepted) doi: 10.1111/jeu.12369-4863. Link: http://onlinelibrary.wiley.com/doi/10.1111/jeu.12369/full
The genus Tetrahymena (Ciliophora, Oligohymenophorea) probably represents the best studied ciliate genus. At present, more than forty species have been described. All are colorless, i.e. they do not harbor symbiotic algae, and as aerobes they need at least microaerobic habitats. Here, we present the morphological and molecular description of the first green representative, Tetrahymena utriculariae n. sp., living in symbiosis with endosymbiotic algae identified as Micractinium sp. (Chlorophyta). The full life cycle of the ciliate species is documented, including trophonts and theronts, conjugating cells, resting cysts and dividers. This species has been discovered in an exotic habitat, namely in traps of the carnivorous aquatic plant Utricularia reflexa (originating from Okavango Delta, Botswana). Green ciliates live as commensals of the plant in this anoxic habitat. Ciliates are bacterivorous, however, symbiosis with algae is needed to satisfy cell metabolism but also to gain oxygen from symbionts. When ciliates are cultivated outside their natural habitat under aerobic conditions and fed with saturating bacterial food, they gradually become aposymbiotic. Based on phylogenetic analyses of 18S rRNA and mitochondrial cox1 genes T. utriculariae forms a sister group to Tetrahymena thermophila.
Šimek, K., Pitsch G., Salcher M.M., Sirová, D., Shabarova, T., Adamec L., Posch, T. Ecological traits of a zoochlorellae-bearing Tetrahymena sp. (Ciliophora) living in traps of the carnivorous aquatic plant Utricularia reflexa. J. Eukaryot. Microbiol. (Accepted): doi: 10.1111/jeu.12368-4864. Link: http://onlinelibrary.wiley.com/doi/10.1111/jeu.12368/full
Trap fluid of aquatic carnivorous plants of the genus Utricularia hosts specific microbiomes consisting of commensal pro- and eukaryotes of largely unknown ecology. We examined the characteristics and dynamics of bacteria and the three dominant eukaryotes, i.e. the algae-bearing ciliate Tetrahymena utriculariae (Ciliophora), a green flagellate Euglena agilis (Euglenophyta), and the alga Scenedesmus alternans (Chlorophyta), associated with the traps of Utricularia reflexa. Our study focused on ecological traits and life strategies of the highly abundant ciliate whose biomass by far exceeds that of other eukaryotes and bacteria independent of the trap age. The ciliate was the only bacterivore in the traps, driving rapid turnover of bacterial standing stock. However, given the large size of the ciliate and the cell-specific uptake rates of bacteria we estimated that bacterivory alone would likely be insufficient to support its apparent rapid growth in traps. We suggest that mixotrophy based on algal symbionts contributes significantly to the diet and survival strategy of the ciliate in the extreme (anaerobic, low pH) trap-fluid environment. We propose a revised concept of major microbial interactions in the trap fluid where ciliate bacterivory plays a central role in regeneration of nutrients bound in rapidly growing bacterial biomass.
Baltar F, Palovaara J, Unrein U, Catala P, Horňák K, Šimek K, Vaqué D, Massana R, Gasol JM, Pinhassi J. 2016: Marine bacterial community structure resilience to changes in protist predation under phytoplankton bloom conditions. ISME J. 10: 568-581. doi:10.1038/ismej.2015.135
To test whether protist grazing selectively affects the composition of aquatic bacterial communities, we combined high-throughput sequencing to determine bacterial community composition with analyses of grazing rates, protist and bacterial abundances and bacterial cell sizes and physiological states in a mesocosm experiment in which nutrients were added to stimulate a phytoplankton bloom. A large variability was observed in the abundances of bacteria (from 0.7 to 2.4 × 106cells per ml), heterotrophic nanoflagellates (from 0.063 to 2.7 × 104 cells per ml) and ciliates (from 100 to 3000 cells per l) during the experiment (∼3-, 45- and 30-fold, respectively), as well as in bulk grazing rates (from 1 to 13 × 106 bacteria per ml per day) and bacterial production (from 3 to 379 μg per C l per day) (1 and 2 orders of magnitude, respectively). However, these strong changes in predation pressure did not induce comparable responses in bacterial community composition, indicating that bacterial community structure was resilient to changes in protist predation pressure. Overall, our results indicate that peaks in protist predation (at least those associated with phytoplankton blooms) do not necessarily triggersubstantial changes in the composition of coastal marine bacterioplankton communities.
Salcher MM, Ewert C, Šimek K, Kasalický V, Posch T. 2016: Interspecific competition and protistan grazing affect the coexistence of freshwater betaproteobacterial strains. FEMS Microb. Ecol. 92 (2), fiv156 doi:10.1093/femsec/fiv156.
The competitive strength of four cosmopolitan freshwater betaproteobacterial isolates was investigated in the presence or absence of bacterivorous flagellates during continuous cultivation in artificial minimal medium at two dilution rates. Bacteria reached similar abundance and growth rate in monocultures, but in co-cultures, two strains (Acidovorax sp. and Massilia sp.) displayed significantly higher numbers and growth rates. These potential cross-feeding benefits were also supported by a high nutritional versatility of the two strains. In contrast,Hydrogenophaga sp. was seemingly less competitive or even inhibited by co-cultivation, and Limnohabitans planktonicusdisplayed striking abundance fluctuations. The latter two strains were least versatile in the uptake of different carbon sources and thus suffered more from interspecific competition. Moreover, remarkable strain-specific responses appeared when bacteria experienced increasing loss rates due to grazing and/or raised dilution rates. Limnohabitans planktonicusdeveloped no successful defence strategy and was close to extinction. Massilia sp. formed grazing-resistant filaments exclusively at low dilution, but was highly reduced at increased flow-through. Acidovorax sp. was selectively ingested, but compensated grazing losses with accelerated growth rates and formed (co-)aggregates together with Hydrogenophagasp. to escape predation at high flow-through. These species-specific interactions, growth responses and defence strategies strongly modulate mixed microbial assemblages and the microbial food web.
Hahn M.W., Jezberová J., Koll U., Saueressig-Beck T., Schmidt J. (2016): Complete ecological isolation and cryptic diversity in Polynucleobacter bacteria not resolved by 16S rRNA gene sequences. The ISME Journal 10:1642-1655. DOI:10.1038/ismej.2015.237
Neuenschwander SM, Salcher MM, Pernthaler J. 2015: Fluorescence in situ hybridization and sequential catalysed reporter deposition (2C-FISH) for the flow cytometric sorting of freshwater ultramicrobacteria. Frontiers in Microbiology 6: 247. DOI:10.3389/fmicb.2015.00247
Flow cytometric sorting is a powerful tool to physically separate cells within mixed microbial communities. If combined with phylogenetic staining (fluorescence in situ hybridization, FISH) it allows to specifically sort defined genotypic microbial populations from complex natural samples. However, the targeted enrichment of freshwater ultramicrobacteria, such as members of the LD12 clade of Alphaproteobacteria (SAR11-IIIb), is still challenging. Current FISH protocols, even in combination with signal amplification by catalysed reporter deposition (CARD), are not sufficiently sensitive for the distinction of these bacteria from background noise by flow cytometry, presumably due to their low ribosome content and small cell sizes. We, therefore, modified a CARD based flow sorting protocol with the aim of increasing its sensitivity to a level sufficient for ultramicrobacteria. This was achieved by a second signal amplification step mediated by horseradish peroxidase labelled antibodies targeted to the fluorophores that were previously deposited by CARD-FISH staining. The protocol was tested on samples from an oligo-mesotrophic lake. Ultramicrobacteria affiliated with LD12 Alphaproteobacteria could be successfully sorted to high purity by flow cytometry. The ratios of median fluorescence signal to background ranged around 20, and hybridization rates determined by flow cytometry were comparable to those obtained by fluorescence microscopy. Potential downstream applications of our modified cell staining approach range from the analysis of microdiversity within 16S rRNA-defined populations to that of functional properties, such as the taxon-specific incorporation rates of organic substrates.
Bohunická M, Mareš J, Hrouzek P, Urajová P, Lukeš M, Šmarda J, Komárek J, Gaysina LA, Strunecký O. 2015: A combined morphological, ultrastructural, molecular, and biochemical study of the peculiar family Gomontiellaceae (Oscillatoriales) reveals a new cylindrospermopsin-producing clade of cyanobacteria. Journal of Phycology: accepted. doi: 10.1111/jpy.12354-15-131.
Members of the morphologically unusual cyanobacterial family Gomontiellaceae were studied using a polyphasic approach. Cultured strains of Hormoscilla pringsheimii, Starria zimbabweënsis, Crinalium magnum, and Crinalium epipsammum were thoroughly examined, and the type specimen of the family, Gomontiella subtubulosa, was investigated. The results of morphological observations using both light microscopy and transmission electron microscopy were consistent with previous reports and provided evidence for the unique morphological and ultrastructural traits of this family. Analysis of the 16S rRNA gene confirmed the monophyletic origin of non-marine representatives of genera traditionally classified into this family. The family was phylogenetically placed among other groups of filamentous cyanobacterial taxa. The presence of cellulose in the cell wall was analyzed and confirmed in all cultured Gomontiellaceae members using Fourier transform infrared spectroscopy and fluorescence microscopy. Evaluation of toxins produced by the studied strains revealed the hepatotoxin cylindrospermopsin (CYN) in available strains of the genus Hormoscilla. Production of this compound in both Hormoscilla strains was detected using HPLC-HRMS and confirmed by positive PCR amplification of the cyrJ gene from the CYN biosynthetic cluster. To our knowledge, this is the first report of CYN production by soil cyanobacteria, establishing a previously unreported CYN- producing lineage. The present study indicates that cyanobacteria of the family Gomontiellaceae form a separate but coherent cluster defined by numerous intriguing morphological, ultrastructural, and biochemical features, and exhibiting a toxic potential worthy of further investigation.