Root Primordium Defective 1 Encodes an Essential PORR Protein Required for the Splicing of Mitochondria Encoded Group II Introns and for Respiratory Complex I Biogenesis.

Cellular respiration involves complex organellar metabolic activities that are pivotal for plant growth and development. Mitochondria contain their own genetic system (mitogenome, mtDNA), which encodes key elements of the respiratory machinery. Plant mtDNAs are notably larger than their counterparts in Animalia, with complex genome organization and gene-expression characteristics. The maturation of the plant mitochondrial transcripts involves extensive RNA editing, trimming and splicing events. These essential processing steps rely on the activities of numerous nuclear-encoded cofactors, which may also play key regulatory roles in mitochondrial biogenesis and function, and hence in plant physiology. Proteins that harbor the Plant Organelle RNA Recognition (PORR) domain are represented in a small gene family in plants. Several PORR members, including WTF1, WTF9 and LEFKOTHEA, are known to act in the splicing of organellar group II introns in angiosperms. The AT4G33495 gene-locus encodes an essential PORR-protein in Arabidopsis, termed as ROOT PRIMORDIUM DEFECTIVE 1 (RPD1). A null mutation of At.RPD1 causes arrest in early embryogenesis, while the missense mutant lines, rpd1.1 and rpd1.2, exhibit a strong impairment in root development and retarded growth phenotypes, especially under high-temperature conditions. Here, we further show that RPD1 functions in the splicing of introns that reside in the coding regions of various complex I (CI) subunits (i.e., nad2, nad4, nad5 and nad7), as well as in the maturation of the ribosomal rps3 pre-RNA in Arabidopsis mitochondria. The altered growth and developmental phenotypes and modified respiration activities are tightly correlated with respiratory chain CI defects in rpd1 mutants.

Anti-lung cancer properties of cyanobacterial bioactive compounds.

Lung cancer, the most prevalent gender-independent tumor entity in both men and women, is among the leading cause of cancer-related deaths worldwide. Despite decades of effort in developing improved therapeutic strategies including immunotherapies and novel chemotherapeutic agents, only modest improvements in outcome and long-term survival of lung cancer patients have been achieved. Therefore, exploring new and exceptional sources for bioactive compounds that might serve as anti-cancer agents might be the key to improving lung cancer therapy. On account of diverse forms, cyanobacteria might serve as a potential source for compounds with potential therapeutic applicability against malignant disorders, including cancer. The assorted arrays of metabolic mechanisms synthesize a plethora of bioactive compounds with immense biological potential. These compounds have been proven to be effective against various cancer cell lines and xenograft animal models. The present review provides an overview of the most promising cyanobacteria-derived bioactive compounds proven to exhibit anti-cancer properties in in-vitro and in-vivo studies and highlights their applicability as potential therapeutic agents with a focus on their anti-lung cancer properties.

Flow cytometric sorting of loricate choanoflagellates from the oligotrophic ocean.

It is challenging to study protists with extensive, loosely-associated extracellular structures because of the problems with keeping specimens intact. Here we have tested the suitability of high-speed flow cytometric sorting as a tool for studying such protists using oceanic loricate choanoflagellates as a model. We chose choanoflagellates because their lorica-to-cell volume ratio is > 10 and the voluminous loricae, i.e., the siliceous cell baskets essential for taxonomic identification, only loosely enclose the cells. Besides, owing to low concentrations, choanoflagellates are grossly under-sampled in the oligotrophic ocean. On four research cruises the small heterotrophic protists from samples collected in the photic layer of the South Atlantic and South Pacific oligotrophic (sub)tropical gyres and adjacent mesotrophic waters were flow sorted at sea for electron microscopy ashore. Among the flow-sorted protozoa we were able to select loricate choanoflagellates to assess their species diversity and concentrations. The well-preserved loricae of flow-sorted choanoflagellates made identification of 29 species from 14 genera possible. In the oligotrophic waters, we found neither endemic species nor evident morphological adaptations other than a tendency for lighter silicification of loricae. Common sightings of specimens storing extra costae in preparation for division, indicate choanoflagellates thriving in oligotrophic waters rather than enduring them. Thus, this case study demonstrates that high-speed flow sorting can assist in studying protists with extracellular structures 16-78× bigger than the enclosed cell.

Notable predominant morphology of the smallest most abundant protozoa of the open ocean revealed by electron microscopy.

In the microbe-driven ecosystems of the open ocean, the small heterotrophic flagellates (sHF) are the chief microbial predators and recyclers of essential nutrients to phototrophic microbes. Even with intensive molecular phylogenetic studies of the sHF, the origins of their feeding success remain obscure because of limited understanding of their morphological adaptations to feeding. Here, we examined the sHF morphologies in the largest, most oligotrophic South Pacific and Atlantic (sub)tropical gyres and adjacent mesotrophic waters. On four research cruises, the sHF cells were flow cytometrically sorted from bacterioplankton and phytoplankton for electron microscopy. The sorted sHF comprised chiefly heterokont (HK) biflagellates and unikont choanoflagellates numerically at around 10-to-1 ratio. Of the four differentiated morphological types of HK omnipresent in the open ocean, the short-tinsel heterokont (stHK), whose tinsel flagellum is too short to propagate a complete wave, is predominant and a likely candidate to be the most abundant predator on Earth. Modeling shows that the described stHK propulsion is effective in feeding on bacterioplankton cells at low concentrations; however, owing to general prey scarcity in the oligotrophic ocean, selective feeding is unsustainable and omnivory is equally obligatory for the seven examined sHF types irrespective of their mode of propulsion.

Loricate choanoflagellates (Acanthoecida) from warm water seas. VII. Calotheca Thomsen and Moestrup, Stephanacantha Thomsen and Syndetophyllum Thomsen and Moestrup.

The tectiform loricate choanoflagellate genera Calotheca, Stephanacantha and Syndetophyllum have all been first described from warm water habitats and share the presence of flattened and often elaborate costal strips in the lorica. The current reinvestigation does confirm both the widespread occurrence of these taxa within the global warm water belt, and largely corroborates the established genus and species matrix. We describe here Stephanacantha oceanica sp. nov. which closely resembles S. campaniformis, and transfer Parvicorbicula zigzag to the genus Stephanacantha, despite differences in costal strip morphology, but based on a complete agreement in lorica constructional details.

Loricate choanoflagellates (Acanthoecida) from warm water seas. VI. Pleurasiga Schiller and Parvicorbicula Deflandre.

The loricate choanoflagellate genera Pleurasiga and Parvicorbicula are taxonomically ambiguous. Pleurasiga because of the uncertainty that relates to the true identity of the type species, and Parvicorbicula because too many newly described species over time have been dumped here in lack of better options. While all species currently allocated to the genus Pleurasiga (with the exception of the type species) are observed in our samples from the global warm water belt, the genus Parvicorbicula is represented by just a few and mostly infrequently recorded taxa. Two new species, viz. Pl. quadrangiella sp. nov. and Pl. minutissima sp. nov., are described here. While the former is closely related to Pl. echinocostata, the latter is reminiscent of Pl. minima. Core species of Pleurasiga and Parvicorbicula deviate from the vast majority of loricate choanoflagellates in having both the anterior and the mid-lorica transverse costae located exterior to the longitudinal costae. In Pl. quadrangiella there is no mid-lorica transverse costa but rather a small posterior transverse costa located inside the longitudinal costae. In Pl. minutissima the mid-lorica transverse costa has extensive costal strip overlaps which reveal patterns of costal strip junctions that deviate from the norm.

Accumulation of ambient phosphate into the periplasm of marine bacteria is proton motive force dependent.

Bacteria acquire phosphate (Pi) by maintaining a periplasmic concentration below environmental levels. We recently described an extracellular Pi buffer which appears to counteract the gradient required for Pi diffusion. Here, we demonstrate that various treatments to outer membrane (OM) constituents do not affect the buffered Pi because bacteria accumulate Pi in the periplasm, from which it can be removed hypo-osmotically. The periplasmic Pi can be gradually imported into the cytoplasm by ATP-powered transport, however, the proton motive force (PMF) is not required to keep Pi in the periplasm. In contrast, the accumulation of Pi into the periplasm across the OM is PMF-dependent and can be enhanced by light energy. Because the conventional mechanism of Pi-specific transport cannot explain Pi accumulation in the periplasm we propose that periplasmic Pi anions pair with chemiosmotic cations of the PMF and millions of accumulated Pi pairs could influence the periplasmic osmolarity of marine bacteria.

Bacterioplankton reveal years-long retention of Atlantic deep-ocean water by the Tropic Seamount.

Seamounts, often rising hundreds of metres above surrounding seafloor, obstruct the flow of deep-ocean water. While the retention of deep-water by seamounts is predicted from ocean circulation models, its empirical validation has been hampered by large scale and slow rate of the interaction. To overcome these limitations we use the growth of planktonic bacteria to assess the retention time of deep-ocean water by a seamount. The selected Tropic Seamount in the North-Eastern Atlantic is representative for the majority of isolated seamounts, which do not affect the surface ocean waters. We prove deep-water is retained by the seamount by measuring 2.4× higher bacterial concentrations in the seamount-associated or 'sheath'-water than in deep-ocean water unaffected by seamounts. Genomic analyses of flow-sorted, dominant sheath-water bacteria confirm their planktonic origin, whilst proteomic analyses of the sheath-water bacteria, isotopically labelled in situ, indicate their slow growth. According to our radiotracer experiments, it takes the sheath-water bacterioplankton 1.5 years to double their concentration. Therefore, the seamount should retain the deep-ocean water for 1.8 years for the deep-ocean bacterioplankton to grow to the 2.4× higher concentration in the sheath-water. We propose that turbulent mixing of the seamount sheath-water stimulates bacterioplankton growth by increasing cell encounter rate with ambient dissolved organic molecules.

Deconstruction of plant biomass by a Cellulomonas strain isolated from an ultra-basic (lignin-stripping) spring.

Plant material falling into the ultra-basic (pH 11.5-11.9) springs within The Cedars, an actively serpentinizing site in Sonoma County, California, is subject to conditions that mimic the industrial pretreatment of lignocellulosic biomass for biofuel production. We sought to obtain hemicellulolytic/cellulolytic bacteria from The Cedars springs that are capable of withstanding the extreme alkaline conditions wherein calcium hydroxide-rich water removes lignin, making cell wall polysaccharides more accessible to microorganisms and their enzymes. We enriched for such bacteria by adding plant debris from the springs into a synthetic alkaline medium with ground tissue of the biofuel crop switchgrass (Panicum virgatum L.) as the sole source of carbon. From the enrichment culture we isolated the facultative anaerobic bacterium Cellulomonas sp. strain FA1 (NBRC 114238), which tolerates high pH and catabolizes the major plant cell wall-associated polysaccharides cellulose, pectin, and hemicellulose. Strain FA1 in monoculture colonized the plant material and degraded switchgrass at a faster rate than the community from which it was derived. Cells of strain FA1 could be acclimated through subculturing to grow at a maximal concentration of 13.4% ethanol. A strain FA1-encoded ß-1, 4-endoxylanase expressed in E. coli was active at a broad pH range, displaying near maximal activity at pH 6-9. Discovery of this bacterium illustrates the value of extreme alkaline springs in the search for microorganisms with potential for consolidated bioprocessing of plant biomass to biofuels and other valuable bio-inspired products.

Sedimentation of ballasted cells-free EPS in meromictic Fayetteville Green Lake.

Fayetteville Green Lake (FGL) is a recognized, extensively studied present-day model of the stratified Proterozoic ocean. Nonetheless, biomass sedimentation in FGL remains hard to explain: while virtually all sediment pigments belong to photosynthetic sulfur bacteria from a chemocline, the isotopic carbon signature of the bulk organic matter suggests its epilimnetic phytoplankton origin. To explain the epilimnetic origin of sedimented carbon, we studied the dominant Synechococci, isolated from FGL. Here, we present experimental evidence that FGL Synechococci produce copious extracellular polysaccharides (EPS) especially when availability of inorganic carbon (Ci ) is high relative to availability of other macronutrients, for example phosphorus. The accumulating EPS become impregnated with calcium, magnesium, and sodium cations and are released to the environment as ballasted cell coverings. Sedimentation of these cell-free EPS can constitute the bulk of pigment-free organic material in FGL sediment. Because increased availability of Ci specifically stimulates production of EPS and the accumulated EPS adsorb cations and become ballasted, we propose the universal role of cyanobacterial EPS in biomass sedimentation in the high-Ci Paleoproterozoic ocean as well as in modern aquatic systems like FGL.

Impact of ferromanganese ore pollution on phytoplankton CO2 fixation in the surface ocean.

Because ferromanganese polymetallic crusts can become a global resource of valuable elements the ecological impact of seafloor crust mining requires evaluation. Whilst the detrimental impact on deep-ocean benthos is established, experimental evidence about the mining hazard to surface-ocean is sparse. When retrieved, mined crusts can leach elements potentially harmfull to the core oceanic CO2-fixers - phytoplankton. To directly assess the magnitude of this potential hazard at ocean-basin scale, we examine the impact of ore slurry on phytoplankton CO2 fixation along a meridional transect through the South Atlantic Ocean. Within 12 h crust slurry additions caused a 25% decrease of CO2 fixation in the subtropical region and 15% in the temperate-polar region. Such moderate susceptibility of phytoplankton indicates limited release of harmful elements from tested polymetallic powder. Although this implies that environmentally sustainable seafloor mining could be feasible, longer-term complex studies of the mining impact on the surface ocean are required.

High pCO2-induced exopolysaccharide-rich ballasted aggregates of planktonic cyanobacteria could explain Paleoproterozoic carbon burial.

The contribution of planktonic cyanobacteria to burial of organic carbon in deep-sea sediments before the emergence of eukaryotic predators ~1.5 Ga has been considered negligible owing to the slow sinking speed of their small cells. However, global, highly positive excursion in carbon isotope values of inorganic carbonates ~2.22-2.06 Ga implies massive organic matter burial that had to be linked to oceanic cyanobacteria. Here to elucidate that link, we experiment with unicellular planktonic cyanobacteria acclimated to high partial CO2 pressure (pCO2) representative of the early Paleoproterozoic. We find that high pCO2 boosts generation of acidic extracellular polysaccharides (EPS) that adsorb Ca and Mg cations, support mineralization, and aggregate cells to form ballasted particles. The down flux of such self-assembled cyanobacterial aggregates would decouple the oxygenic photosynthesis from oxidative respiration at the ocean scale, drive export of organic matter from surface to deep ocean and sustain oxygenation of the planetary surface.

"Pomacytosis"-Semi-extracellular phagocytosis of cyanobacteria by the smallest marine algae.

The smallest algae, less than 3 µm in diameter, are the most abundant eukaryotes of the World Ocean. Their feeding on planktonic bacteria of similar size is globally important but physically enigmatic. Tiny algal cells tightly packed with the voluminous chloroplasts, nucleus, and mitochondria appear to have insufficient organelle-free space for prey internalization. Here, we present the first direct observations of how the 1.3-µm algae, which are only 1.6 times bigger in diameter than their prey, hold individual Prochlorococcus cells in their open hemispheric cytostomes. We explain this semi-extracellular phagocytosis by the cell size limitation of the predatory alga, identified as the Braarudosphaera haptophyte with a nitrogen (N2)-fixing endosymbiont. Because the observed semi-extracellular phagocytosis differs from all other types of protistan phagocytosis, we propose to name it "pomacytosis" (from the Greek πώµα for "plug").

Installing extra bicarbonate transporters in the cyanobacterium Synechocystis sp. PCC6803 enhances biomass production.

As a means to improve carbon uptake in the cyanobacterium Synechocystis sp. strain PCC6803, we engineered strains to contain additional inducible copies of the endogenous bicarbonate transporter BicA, an essential component of the CO2-concentrating mechanism in cyanobacteria. When cultured under atmospheric CO2 pressure, the strain expressing extra BicA transporters (BicA(+) strain) grew almost twice as fast and accumulated almost twice as much biomass as the control strain. When enriched with 0.5% or 5% CO2, the BicA(+) strain grew slower than the control but still showed a superior biomass production. Introducing a point mutation in the large C-terminal cytosolic domain of the inserted BicA, at a site implicated in allosteric regulation of transport activity, resulted in a strain (BicA(+)(T485G) strain) that exhibited pronounced cell aggregation and failed to grow at 5% CO2. However, the bicarbonate uptake capacity of the induced BicA(+)(T485G) was twice higher than for the wild-type strain. Metabolic analyses, including phenotyping by synchrotron-radiation Fourier transform Infrared spectromicroscopy, scanning electron microscopy, and lectin staining, suggest that the excess assimilated carbon in BicA(+) and BicA(+)(T485G) cells was directed into production of saccharide-rich exopolymeric substances. We propose that the increased capacity for CO2 uptake in the BicA(+) strain can be capitalized on by re-directing carbon flux from exopolymeric substances to other end products such as fuels or high-value chemicals.

Characterization of cyanate metabolism in marine Synechococcus and Prochlorococcus spp.

Cyanobacteria of the genera Synechococcus and Prochlorococcus are the most abundant photosynthetic organisms on earth, occupying a key position at the base of marine food webs. The cynS gene that encodes cyanase was identified among bacterial, fungal, and plant sequences in public databases, and the gene was particularly prevalent among cyanobacteria, including numerous Prochlorococcus and Synechococcus strains. Phylogenetic analysis of cynS sequences retrieved from the Global Ocean Survey database identified >60% as belonging to unicellular marine cyanobacteria, suggesting an important role for cyanase in their nitrogen metabolism. We demonstrate here that marine cyanobacteria have a functionally active cyanase, the transcriptional regulation of which varies among strains and reflects the genomic context of cynS. In Prochlorococcus sp. strain MED4, cynS was presumably transcribed as part of the cynABDS operon, implying cyanase involvement in cyanate utilization. In Synechococcus sp. strain WH8102, expression was not related to nitrogen stress responses and here cyanase presumably serves in the detoxification of cyanate resulting from intracellular urea and/or carbamoyl phosphate decomposition. Lastly, we report on a cyanase activity encoded by cynH, a novel gene found in marine cyanobacteria only. The presence of dual cyanase genes in the genomes of seven marine Synechococcus strains and their respective roles in nitrogen metabolism remain to be clarified.