Unravelling the microbial role in ooid formation – results of an in situ experiment in modern freshwater Lake Geneva in Switzerland

The microbial role in the formation of the cortex of low‐Mg calcite freshwater ooids in western part of Lake Geneva in Switzerland has been suggested previously, but not demonstrated conclusively. Early work mostly concentrated in hypersaline milieus, and hence little is known about their genesis in freshwater environments. We designed an in situ experiment to mimic the natural process of low‐Mg calcite precipitation. A special device was placed in the ooid‐rich bank of the lake. It contained frosted glass (SiO₂) slides, while quartz (SiO₂) is the most abundant mineral composition of ooid nuclei that acted as artificial substrates to favour microbial colonization. Microscopic inspection of the slides revealed a clear seasonal pattern of carbonate precipitates, which were always closely associated with biofilms that developed on the surface of the frosted slides containing extracellular polymeric substance, coccoid and filamentous cyanobacteria, diatoms and heterotrophic bacteria. Carbonate precipitation peaks during early spring and late summer, and low‐Mg calcite crystals mostly occur in close association with filamentous and coccoid cyanobacteria (e.g. Tolypothrix, Oscillatoria and Synechococcus, Anacystis, respectively). Further scanning electron microscope inspection of the samples revealed low‐Mg calcite with crystal forms varying from anhedral to euhedral rhombohedra, depending on the seasons. Liquid cultures corroborate the in situ observations and demonstrate that under the same physicochemical conditions the absence of biofilms prevents the precipitation of low‐Mg calcite crystals. These results illustrate that biofilms play a substantial role in low‐Mg calcite ooid cortex formation. It further demonstrates the involvement of microbes in the early stages of ooid development. Combined with ongoing microbial cultures under laboratory‐controlled conditions, the outcome of our investigation favoured the hypothesis of external microbial precipitation of low‐Mg calcite as the main mechanism involved in the early stage of ooid formation in freshwater Lake Geneva.     

GROWTH AND CARBON CONTENT OF THREE DIFFERENT‐SIZED DIAZOTROPHIC CYANOBACTERIA OBSERVED IN THE SUBTROPICAL NORTH PACIFIC1

To develop tools for modeling diazotrophic growth in the open ocean, we determined the maximum growth rate and carbon content for three diazotrophic cyanobacteria commonly observed at Station ALOHA (A Long‐term Oligotrophic Habitat Assessment) in the subtropical North Pacific: filamentous nonheterocyst‐forming Trichodesmium and unicellular Groups A and B. Growth‐irradiance responses of Trichodesmium erythraeum Ehrenb. strain IMS101 and Crocosphaera watsonii J. Waterbury strain WH8501 were measured in the laboratory. No significant differences were detected between their fitted parameters (±CI) for maximum growth rate (0.51 ± 0.09 vs. 0.49 ± 0.17 d^?1), half‐light saturation (73 ± 29 vs. 66 ± 37 μmol quanta · m^?2 · s^?1), and photoinhibition (0 and 0.00043 ± 0.00087 [μmol quanta · m^?2 · s^?1]^?1). Maximum growth rates and carbon contents of Trichodesmium and Crocosphaera cultures conformed to published allometric relationships, demonstrating that these relationships apply to oceanic diazotrophic microorganisms. This agreement promoted the use of allometric models to approximate unknown parameters of maximum growth rate (0.77 d^?1) and carbon content (480 fg C · μm^?3) for the uncultivated, unicellular Group A cyanobacteria. The size of Group A was characterized from samples from the North Pacific Ocean using fluorescence‐activated cell sorting and real‐time quantitative PCR techniques. Knowledge of growth and carbon content properties of these organisms facilitates the incorporation of different types of cyanobacteria in modeling efforts aimed at assessing the relative importance of filamentous and unicellular diazotrophs to carbon and nitrogen cycling in the open ocean.     

Measuring carbon and N2 fixation in field populations of colonial and free‐living unicellular cyanobacteria using nanometer‐scale secondary ion mass spectrometry1

Unicellular cyanobacteria are now recognized as important to the marine N and C cycles in open ocean gyres, yet there are few direct in situ measurements of their activities. Using a high‐resolution nanometer scale secondary ion mass spectrometer (nanoSIMS), single cell N₂ and C fixation rates were estimated for unicellular cyanobacteria resembling N₂ fixer Crocosphaera watsonii. Crocosphaera watsonii‐like cells were observed in the subtropical North Pacific gyre (22°45′ N, 158°0′ W) as 2 different phenotypes: colonial and free‐living. Colonies containing 3–242 cells per colony were observed and cell density in colonies increased with incubation time. Estimated C fixation rates were similarly high in both phenotypes and unexpectedly for unicellular cyanobacteria 85% of the colonial cells incubated during midday were also enriched in ¹⁵N above natural abundance. Highest ¹⁵N enrichment and N₂ fixation rates were found in cells incubated overnight where up to 64% of the total daily fixed N in the upper surface waters was attributed to both phenotypes. The colonial cells retained newly fixed C in a sulfur‐rich matrix surrounding the cells and often cells of both phenotypes possessed areas (<1 nm) of enriched ¹⁵N and ¹³C resembling storage granules. The nanoSIMS imaging of the colonial cells also showed evidence for a division of N₂ and C fixation activity across the colony where few individual cells (<34%) in a given colony were enriched in both ¹⁵N and ¹³C above the colony average. Our results provide new insights into the ecophysiology of unicellular cyanobacteria.     

SIDEROPHORE‐INDEPENDENT IRON UPTAKE BY IRON‐LIMITED CELLS OF THE CYANOBACTERIUM ANABAENA FLOS‐AQUAE1

Iron acquisition by iron‐limited cyanobacteria is typically considered to be mediated mainly by siderophores, iron‐chelating molecules released by iron‐limited cyanobacteria into the environment. In this set of experiments, iron uptake by iron‐limited cells of the cyanobacterium Anabaena flos‐aquae (L.) Bory was investigated in cells resuspended in siderophore‐free medium. Removal of siderophores decreased iron‐uptake rates by ～60% compared to siderophore‐replete conditions; however, substantial rates of iron uptake remained. In the absence of siderophores, Fe(III) uptake was much more rapid from a weaker synthetic chelator [N‐(2‐hydroxyethyl)ethylenediamine‐N,N′,N′‐triacetic acid (HEDTA); log K_cond = 28.64 for Fe(III)HEDTA(OH)^?] than from a very strong chelator [N,N′‐bis(2‐hydroxybenzyl)‐ethylenediamine‐N,N′‐diacetic acid (HBED); log K_cond = 31.40 for Fe(III)HBED^?], and increasing chelator:Fe(III) ratios decreased the Fe(III)‐uptake rate; these results were evident in both short‐term (4 h; absence of siderophores) and long‐term (116 h; presence of siderophores) experiments. However, free (nonchelated) Fe(III) provided the most rapid iron uptake in siderophore‐free conditions. The results of the short‐term experiments are consistent with an Fe(III)‐binding/uptake mechanism associated with the cyanobacterial outer membrane that operates independently of extracellular siderophores. Iron uptake was inhibited by temperature‐shock treatments of the cells and by metabolically compromising the cells with diphenyleneiodonium; this finding indicates that the process is dependent on active metabolism to operate and is not simply a passive Fe(III)‐binding mechanism. Overall, these results point to an important, siderophore‐independent iron‐acquisition mechanism by iron‐limited cyanobacterial cells.     

Identification of glycine betaine as compatible solute in Synechococcus sp. WH8102 and characterization of its N-methyltransferase genes involved in betaine synthesis

Biosynthesis of glycine betaine from simple carbon sources as compatible solute is rare among aerobic heterotrophic eubacteria, and appears to be almost exclusive to the non-halophilic and slightly halophilic phototrophic cyanobacteria. Although Synechococcus sp. WH8102 (CCMP2370), a unicellular marine cyanobacterium, could grow up to additional 2.5% (w/v) NaCl in SN medium, natural abundance ¹³C nuclear magnetic resonance spectroscopy identified glycine betaine as its major compatible solute. Intracellular glycine betaine concentrations were dependent on the osmolarity of the growth medium over the range up to additional 2% NaCl in SN medium, increasing from 6.8 ± 1.5 to 62.3 ± 5.5 mg/g dw. The ORFs SYNW1914 and SYNW1913 from Synechococcus sp. WH8102 were found as the homologous genes coding for glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase, heterologously over-expressed respectively as soluble fraction in Escherichia coli BL21(DE3)pLysS and purified by Ni-NTA His•bind resins. Their substrate specificities and the values of the kinetic parameters were determined by TLC and ¹H NMR spectroscopy. RT-PCR analysis revealed that the two ORFs were both transcribed in cells of Synechococcus sp. WH8102 growing in SN medium without additional NaCl, which confirmed the pathway of de novo synthesizing betaine from glycine existing in these marine cyanobacteria.     

Different organization of nif genes in nonheterocystous and heterocystous cyanobacteria

Summary Labeled probes carrying the Anabaena PCC 7120 nitrogenase (nifK and nifD) and nitrogenase reductase (nifH) genes were hybridized to Southern blots of DNA from diverse N₂-fixing cyanobacteria in order to test a previous observation of different nif gene organization in nonheterocystous and heterocystous strains. The nif probes showed no significant hybridization to DNA from a unicellular cyanobacterium incapable of N₂ fixation. All nonheterocystous cyanobacteria examined (unicellular and filamentous) had a contiguous nifKDH gene cluster whereas all of the heterocystous strains showed separation of nifK from contiguous nifDH genes. These findings suggest that nonheterocystous and heterocystous cyanobacteria have characteristic and fundamentally different nif gene arrangements. The noncontiguous nif gene pattern, as shown with two Het^- mutants, is independent of phenotypic expression of heterocyst differentiation and aerobic N₂-fixation. Thus nif arrangement could be a useful taxonomic marker to distinguish between phenotypically Het^- heterocystous cyanobacteria and phylogenetically unrelated nonheterocystous strains.     

CHARACTERIZATION OF A FUNCTIONAL VANADIUM‐DEPENDENT BROMOPEROXIDASE IN THE MARINE CYANOBACTERIUM SYNECHOCOCCUS SP. CC93111

Vanadium‐dependent bromoperoxidases (VBPOs) are characterized by the ability to oxidize halides using hydrogen peroxide. These enzymes are well‐studied in eukaryotic macroalgae and are known to produce a variety of brominated secondary metabolites. Though genes have been annotated as VBPO in multiple prokaryotic genomes, they remain uncharacterized. The genome of the coastal marine cyanobacterium Synechococcus sp. CC9311 encodes a predicted VBPO (YP_731869.1, sync_2681), and in this study, we show that protein extracts from axenic cultures of Synechococcus possess bromoperoxidase activity, oxidizing bromide and iodide, but not chloride. In‐gel activity assays of Synechococcus proteins separated using PAGE reveal a single band having VBPO activity. When sequenced via liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS), peptides from the band aligned to the VBPO sequence predicted by the open reading frame (ORF) sync_2681. We show that a VBPO gene is present in a closely related strain, Synechococcus sp. WH8020, but not other clade I Synechococcus strains, consistent with recent horizontal transfer of the gene into Synechococcus. Diverse cyanobacterial‐like VBPO genes were detected in a pelagic environment off the California coast using PCR. Investigation of functional VBPOs in unicellular cyanobacteria may lead to discovery of novel halogenated molecules and a better understanding of these organisms’ chemical ecology and physiology.     

A freshwater analog for the production of Epiphyton‐like microfossils

Calcified microbial microfossils—often interpreted as cyanobacteria—were important components of Precambrian and Paleozoic limestones, but their paucity in modern marine environments complicates our ability to make conclusive interpretations about their taxonomic affinity and geologic significance. Freshwater spring‐associated limestones (e.g., travertine and tufa) serve as terrestrial analogs to investigate mineralization in and around aquatic biofilms on observable timescales. We document the diagenesis of calcite fabrics associated with the freshwater algae Oocardium stratum, an epiphytic colonial green algae (desmid) known for producing stalks of extracellular polymeric substances (EPS) and passively producing a bifurcating tubular calcite monocrystal. Bifurcating EPS stalks produced by Oocardium colonies can become calcified and preserved in ancient carbonate deposits. Calcified micritic EPS stalks have a filamentous morphology, show evidence of branching, and maintain uniformity in diameter thickness throughout the mm‐scale colony, much like the enigmatic calcimicrobe Epiphyton. We provide a mechanism by which calcification associated with a colonial semispherical micro‐organism produces microfossils that deceptively resemble filamentous forms. These findings have implications for the use of morphological traits when assigning taxonomic affinities to extinct microfossil groups and highlight the utility of calcifying freshwater modern environments to investigate microbial taphonomy.     

Daphnia Pre‐Exposed to Toxic Microcystis Exhibit Feeding Selectivity

In this study, short term feeding experiments were used to determine if Daphnia, pre‐exposed to cyanobacteria, could avoid cyanobacteria through feeding selectively. The results show that Daphnia ambigua were capable of altering the proportion of a single – celled cyanobacterium (Microcystis aeruginosa) to a chlorophyte (Chlorella kessleri) of similar size and shape. Also, D. ambigua pre‐exposed for eight weeks to a toxic strain of M. aeruginosa were more successful at avoiding this strain than D. ambigua pre‐exposed to C. kessleri or to a non‐toxic strain of M. aeruginosa. These experimental results suggest that further investigations of Daphnia 's ability to influence the dominance of cyanobacterial strains in lakes are warranted. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Palaeoecology of a billion‐year‐old non‐marine cyanobacterium from the Torridon Group and Nonesuch Formation

A new chroococcalean cyanobacterium is described from approximately 1‐billion‐year‐old non‐marine deposits of the Torridonian Group of Scotland and the Nonesuch Formation of Michigan, USA. Individual cells of the new microfossil, Eohalothece lacustrina gen. et sp. nov., are associated with benthic microbial biofilms, but the majority of samples are recovered in palynological preparations in the form of large, apparently planktonic colonies, similar to extant species of Microcystis. In the Torridonian, Eohalothece is associated with phosphatic nodules, and we have developed a novel hypothesis linking Eohalothece to phosphate deposition in ancient freshwater settings. Extant cyanobacteria can be prolific producers of extracellular microcystins, which are non‐ribosomal polypeptide phosphatase inhibitors. Microcystins may have promoted the retention and concentration of sedimentary organic phosphate prior to mineralization of francolite and nodule formation. This has a further implication that the Torridonian lakes were nitrogen limited as the release of microcystins is enhanced under such conditions today. The abundance and wide distribution of Eohalothece lacustrina attests to the importance of cyanobacteria as oxygen‐producing photoautotrophs in lacustrine ecosystems at the time of the Mesoproterozoic–Neoproterozoic transition.     

Identification of inner membrane translocase components of TolC‐mediated secretion in the cyanobacterium Synechocystis sp. PCC 6803

Cyanobacteria were the first organisms ever to perform oxygenic photosynthesis and still significantly contribute to primary production on a global scale. To assure the proper functioning of their primary metabolism and cell homeostasis, cyanobacteria must rely on efficient transport systems to cross their multilayered cell envelope. However, cyanobacterial secretion mechanisms remain largely unknown. Here, we report on the identification of 11 putative inner membrane translocase components of TolC‐mediated secretion in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Gene‐inactivation of each of the candidate genes followed by a comprehensive phenotypic characterization allowed to link specific protein components to the processes of protein export (as part of the type I secretion system) and drug efflux (part of the resistance‐division‐nodulation efflux pumps). In addition, mutants in genes sll0141, sll0180 and slr0369 exhibited alterations in pilin glycosylation, but pili structures could still be observed by transmission electron microscopy. By studying the release of outer membrane vesicles (OMVs), an alternative secretion route, on mutants with impaired secretory functions we suggest that the hyper‐vesiculating phenotype of the TolC‐deficient mutant is related to cell envelope stress management. Altogether, these findings highlight how both classical (TolC‐mediated) and nonclassical (OMVs‐mediated) secretion systems are crucial for cyanobacterial cell homeostasis.     

The recovery of a very shallow eutrophic lake, 20 years after the control of effluent derived phosphorus

1. Monitoring at fortnightly to monthly intervals of a very shallow, lowland lake over 24 years has enabled the time course of recovery from nutrient enrichment to be investigated after high external P loading of the lake (>10 g P m^?2 year^?1) was reduced between 1977 and 1980. 2. The lake showed a relatively rapid response during the spring and early summer, with a reduction in phytoplankton biomass occurring after 5 years when soluble reactive phosphorus concentration was <10 μg L^?1. 3. However, during the later summer the response was delayed for 15 years because of sustained remobilisation of phosphorus from the sediment. The greater water clarity in spring and a gradual shift from planktonic to benthic algal growth may be related to the reduction in internal loading after 15 years. 4. Changes in the phytoplankton community composition were also observed. Centric diatoms became less dominant in the spring, and the summer cyanobacteria populations originally dominated by non‐heterocystous species (Limnothrix/Planktothrix spp.) almost disappeared. Heterocystous species (Anabaena spp. and Aphanizomenon flos‐aquae) were slower to decline, but after 20 years the phytoplankton community was no longer dominated by cyanobacteria. 5. There were no substantial changes in food web structure following re‐oligotrophication. Total zooplankton biomass decreased but body size of Daphnia hyalina, the largest zooplankton species in the lake, remained unchanged, suggesting that the fish population remained dominated by planktivorous species. 6. Macrophyte growth was still largely absent after 20 years, although during the spring water clarity may have become sufficient for macrophytes to re‐establish.     

Oxygen and the light–dark cycle of nitrogenase activity in two unicellular cyanobacteria

Cyanobacteria capable of fixing dinitrogen exhibit various strategies to protect nitrogenase from inactivation by oxygen. The marine Crocosphaera watsonii WH8501 and the terrestrial Gloeothece sp. PCC6909 are unicellular diazotrophic cyanobacteria that are capable of aerobic nitrogen fixation. These cyanobacteria separate the incompatible processes of oxygenic photosynthesis and nitrogen fixation temporally, confining the latter to the dark. Although these cyanobacteria thrive in fully aerobic environments and can be cultivated diazotrophically under aerobic conditions, the effect of oxygen is not precisely known due to methodological limitations. Here we report the characteristics of nitrogenase activity with respect to well‐defined levels of oxygen to which the organisms are exposed, using an online and near real‐time acetylene reduction assay combined with sensitive laser‐based photoacoustic ethylene detection. The cultures were grown under an alternating 12–12 h light–dark cycle and acetylene reduction was recorded continuously. Acetylene reduction was assayed at 20%, 15%, 10%, 7.5%, 5% and 0% oxygen and at photon flux densities of 30 and 76 μmol m^?2 s^?1 provided at the same light–dark cycle as during cultivation. Nitrogenase activity was predominantly but not exclusively confined to the dark. At 0% oxygen nitrogenase activity in Gloeothece sp. was not detected during the dark and was shifted completely to the light period, while C. watsonii did not exhibit nitrogenase activity at all. Oxygen concentrations of 15% and higher did not support nitrogenase activity in either of the two cyanobacteria. The highest nitrogenase activities were at 5–7.5% oxygen. The highest nitrogenase activities in C. watsonii and Gloeothece sp. were observed at 29°C. At 31°C and above, nitrogenase activity was not detected in C. watsonii while the same was the case at 41°C and above in Gloeothece sp. The differences in the behaviour of nitrogenase activity in these cyanobacteria are discussed with respect to their presumed physiological strategies to protect nitrogenase from oxygen inactivation and to the environment in which they thrive.     

A Novel Epiphytic Chlorophyll d‐containing Cyanobacterium Isolated from a Mangrove‐associated Red Alga

A new habitat and a new chlorophyll (Chl) d‐containing cyanobacterium belonging to the genus Acaryochloris are reported in this study. Hyperspectral microscopy showed the presence of Chl d‐containing microorganisms in epiphytic biofilms on a red alga (Gelidium caulacantheum) colonizing the pneumato‐phores of a temperate mangrove (Avicennia marina). The presence of Chl d was further proven by high performance liquid chromatography (HPLC)‐based pigment analysis and by confocal imaging of cultured cells. Enrichment of mangrove biofilm samples under near‐infrared radiation (NIR) yielded the new Acaryochloris sp. MPGRS1, which was closely related in terms of 16S rRNA gene sequence to an isolate from the hypertrophic Salton Sea, USA. The new isolate used Chl d as its major photopigment; Chl d and Chl a contents were ~98% and 1%–2% of total cellular chlorophyll, respectively. These findings expand the variety of ecological niches known to harbor Chl d‐containing cyanobacteria and support our working hypothesis that such oxyphototrophs may be ubiquitous in habitats depleted of visible light, but with sufficient NIR exposure.     

Characterization of the Biocidal Spectrum of Extracellular Filtrates of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis>

The effect of dichloromethane (DCM) and ethyl acetate (EA) extract of a cyanobacterium, Microcystis aeruginosa was evaluated against unicellular cyanobacteria and the phytopathogenic fungus Rhizoctonia solani. Fractionation of the filtrate showed the presence of five spots of different Rf values on silica gel coated plates indicating the presence of a number of compounds in the extract. A marked reduction in growth (52%) of the fungus was recorded on the plates supplemented with cyanobacterial extract, indicating the involvement of anti-fungal metabolite(s). The extract did not show any negative influence on seed germination and growth of seedlings of wheat, rice and mung, emphasizing the suitability of the compound for use in agriculture.     

Application of real-time PCR in the assessment of the toxic cyanobacterium <Emphasis Type="Italic">Cylindrospermopsis raciborskii</Emphasis> abundance and toxicological potential

Cyanobacteria are prokaryotic photosynthetic microorganisms that pose a serious threat to aquatic environments because they are able to form blooms under eutrophic conditions and produce toxins. Cylindrospermopsis raciborskii is a planktonic heterocystous filamentous cyanobacterium initially assigned to the tropics but currently being found in more temperate regions such as Portugal, the southernmost record for this species in Europe. Cylindrospermopsin originally isolated from C. raciborskii is a cytotoxic alkaloid that affects the liver, kidney, and other organs. It has a great environmental impact associated with cattle mortality and human morbidity. Aiming in monitoring this cyanobacterium and its related toxin, a shallow pond located in the littoral center of Portugal, Vela Lake, used for agriculture and recreational purposes was monitored for a 2-year period. To accomplish this, we used the real-time PCR methodology in field samples to quantify the variation of specific genetic markers with primers previously described characterizing total cyanobacteria (16S rRNA), C. raciborskii (rpoC1), and cylindrospermopsin synthetase gene (pks). The results report the high abundance of both cyanobacteria and C. raciborskii in Vela Lake, with C. raciborskii representing 0.4% to 58% of the total cyanobacteria population. Cylindrospermopsin synthetase gene was detected in one of the samples. We believe that with the approach developed in this study, it will be possible to monitor C. raciborskii population dynamics and seasonal variation, as well as the potential toxin production in other aquatic environments.