Jerry J. Brand

Environmental Barcoding Reveals Massive Dinoflagellate Diversity in Marine Environments

Background Dinoflagellates are an ecologically important group of protists with important functions as primary producers, coral symbionts and in toxic red tides. Although widely studied, the natural diversity of dinoflagellates is not well known. DNA barcoding has been utilized successfully for many protist groups. We used this approach to systematically sample known “species”, as a reference to measure the natural diversity in three marine environments. Methodology/Principal Findings In this study, we assembled a large cytochrome c oxidase 1 (COI) barcode database from 8 public algal culture collections plus 3 private collections worldwide resulting in 336 individual barcodes linked to specific cultures. We demonstrate that COI can identify to the species level in 15 dinoflagellate genera, generally in agreement with existing species names. Exceptions were found in species belonging to genera that were generally already known to be taxonomically challenging, such as Alexandrium or Symbiodinium. Using this barcode database as a baseline for cultured dinoflagellate diversity, we investigated the natural diversity in three diverse marine environments (Northeast Pacific, Northwest Atlantic, and Caribbean), including an evaluation of single-cell barcoding to identify uncultivated groups. From all three environments, the great majority of barcodes were not represented by any known cultured dinoflagellate, and we also observed an explosion in the diversity of genera that previously contained a modest number of known species, belonging to Kareniaceae. In total, 91.5% of non-identical environmental barcodes represent distinct species, but only 51 out of 603 unique environmental barcodes could be linked to cultured species using a conservative cut-off based on distances between cultured species. Conclusions/Significance COI barcoding was successful in identifying species from 70% of cultured genera. When applied to environmental samples, it revealed a massive amount of natural diversity in dinoflagellates. This highlights the extent to which we underestimate microbial diversity in the environment.

Isolation of the intact photosystem I reaction center core containing P700 and iron‐sulfur center FX

The photosystem I reaction center core containing P700 and iron‐sulfur center FX has been isolated from a Synechococcus photosystem I particle with 6.8 M urea at pH 10.0 followed by sucrose density ultracentrifugation. The reaction center core has retained > 90% of FX and 100% of P700 (determined by optical spectroscopy) but is totally devoid of iron‐sulfur centers FA and FB (determined by optical and ESR spectroscopy). SDS‐PAGE indicates the retention of the 57 kDa reaction center polypeptide(s) but the total absence of the 16.4 and 8.1 kDa polypeptides. The loss of FA and FB is further reflected in the decline of acid‐labile sulfide from 11.8 ± 0.4 S2− /P700 in the control particle to 4.6 ± 0.3 S2− /P700 in the reaction center core. This preparation represents the first isolation of an intact reaction center core incorporating the components P700 and FX but totally lacking FA and FB.

A unique pseudanabaenalean (cyanobacteria) genus Nodosilinea gen. nov. based on morphological and molecular data

The cyanobacteria are a diverse, ancient lineage of oxygenic, phototrophic bacteria. Ubiquitous in nearly all ecosystems, the alpha‐level diversity of these organisms lags behind other algal lineages due to a perceived dearth of phylogenetically useful characters. Recent phylogenetic studies of species within the genus Leptolyngbya have demonstrated that this is a polyphyletic assemblage. One group of strains that fits within the current circumscription of Leptolyngbya is genetically and phylogenetically distinct from Leptolyngbya sensu stricto. Members of this clade possess both a morphological synapomorphy and shared 16S‐23S internal transcribed spacer (ITS) secondary structure, allowing the diagnosis of the new cyanobacterial genus Nodosilinea. Members of this genus are united by the unique ability to form nodules along the length of the filament. This trait has been previously observed only in the species Leptolyngbya nodulosa Z. Li et J. Brand, and we have chosen this species as the generitype of Nodosilinea. We currently recognize four species in the genus, N. nodulosa (Z. Li et J. Brand) comb. nov., N. bijugata (Kong.) comb. nov., N. conica sp. nov., and N. epilithica sp. nov.

Evidence for direct roles of calcium in photosynthesis

Calcium may function directly in several aspects of photosynthesis. It appears to modulate activity of the phosphatase enzymes in the carbon reduction cycle and also to regulate chloroplast NAD+ kinase activity through a calmodulin-like protein. Some evidence supports a calcium function in the water-splitting complex, and other evidence indicates a reaction center function in photosystem II. Calcium in reaction center II may be tightly bound in chloroplasts and weakly bound in blue-green algal thylakoids. Free calcium concentration in stroma is probably <10−6 M, although the absolute concentration is not yet known. Intrathylakoid calcium content is likely very high. Stromal calcium may regulate several enzyme activities, while intrathylakoid calcium may promote photosystem II constitutively. Results to date demonstrate the need for more attention to cation composition in studies of both light and dark reactions of photosynthesis, and the need to identify free calcium levels in chloroplasts.

Evaluating the Ribosomal Internal Transcribed Spacer (ITS) as a Candidate Dinoflagellate Barcode Marker

Background DNA barcoding offers an efficient way to determine species identification and to measure biodiversity. For dinoflagellates, an ancient alveolate group of about 2000 described extant species, DNA barcoding studies have revealed large amounts of unrecognized species diversity, most of which is not represented in culture collections. To date, two mitochondrial gene markers, Cytochrome Oxidase I (COI) and Cytochrome b oxidase (COB), have been used to assess DNA barcoding in dinoflagellates, and both failed to amplify all taxa and suffered from low resolution. Nevertheless, both genes yielded many examples of morphospecies showing cryptic speciation and morphologically distinct named species being genetically similar, highlighting the need for a common marker. For example, a large number of cultured Symbiodinium strains have neither taxonomic identification, nor a common measure of diversity that can be used to compare this genus to other dinoflagellates. Methodology/Principal Findings The purpose of this study was to evaluate the Internal Transcribed Spacer units 1 and 2 (ITS) of the rDNA operon, as a high resolution marker for distinguishing species dinoflagellates in culture. In our study, from 78 different species, the ITS barcode clearly differentiated species from genera and could identify 96% of strains to a known species or sub-genus grouping. 8.3% showed evidence of being cryptic species. A quarter of strains identified had no previous species identification. The greatest levels of hidden biodiversity came from Scrippsiella and the Pfiesteriaceae family, whilst Heterocapsa strains showed a high level of mismatch to their given species name. Conclusions/Significance The ITS marker was successful in confirming species, revealing hidden diversity in culture collections. This marker, however, may have limited use for environmental barcoding due to paralogues, the potential for unidentifiable chimaeras and priming across taxa. In these cases ITS would serve well in combination with other markers or for specific taxon studies.

Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO2

Photosynthetic microbes are of emerging interest as production organisms in biotechnology because they can grow autotrophically using sunlight, an abundant energy source, and CO2, a greenhouse gas. Important traits for such microbes are fast growth and amenability to genetic manipulation. Here we describe Synechococcus elongatus UTEX 2973, a unicellular cyanobacterium capable of rapid autotrophic growth, comparable to heterotrophic industrial hosts such as yeast. Synechococcus UTEX 2973 can be readily transformed for facile generation of desired knockout and knock-in mutations. Genome sequencing coupled with global proteomics studies revealed that Synechococcus UTEX 2973 is a close relative of the widely studied cyanobacterium Synechococcus elongatus PCC 7942, an organism that grows more than two times slower. A small number of nucleotide changes are the only significant differences between the genomes of these two cyanobacterial strains. Thus, our study has unraveled genetic determinants necessary for rapid growth of cyanobacterial strains of significant industrial potential.

Rapid turnover of a component required for photosynthesis explains temperature dependence and kinetics of photoinhibition in a cyanobacterium, Synechococcus 6301

Illumination of a liquid culture of Synechococcus 6301 at high photon flux density (PFD) elicits a time-dependent first-order exponential decline in relative quantum yield of photosynthetic O2 evolution to some steady-state value. Full photosynthetic activity is restored, also as a time-dependent first-order process, when the photoinhibited culture is transferred to lower PFD. Temperature and irradiation dependence of photoinhibition were measured under conditions which precluded simultaneous recovery from photoinhibition. Also the temperature and irradiation dependence of recovery from photoinhibition were determined under conditions which precluded simultaneous photoinhibition. Kinetics of photoinhibition were sensitive to PFD but relatively independent of temperature. Kinetics of recovery saturated at low PFD but were very temperature dependent at all PFDs. A general equation can be written to predict the change in photosynthetic activity versus time when a cell culture is placed at photoinhibitory PFD, assuming that first-order exponential photoinhibition and first-order exponential recovery from photoinhibition occur simultaneously. The equation can be made specific if the values of the kinetic constant for photoinhibition and for recovery from photoinhibition are known for the particular environmental conditions to which the cells are exposed. These values can be obtained by independently measuring the kinetics of photoinhibition without simultaneous recovery and the kinetics of recovery without simultaneous photoinhibition. The curve of photosynthetic activity versus time for cells placed at high PFD, which is predicted by this equation, precisely fits the experimentally determined kinetics of photoinhibition. This correlation remains valid over a wide range of temperatures and PFDs. Identical results were obtained with the marine cyanobacterium Synechococcus 7002. We conclude that the extent of net photoinhibition over a broad range of conditions represents a sum of individual rates of simultaneous photoinhibition and recovery from photoinhibition. The results support previous proposals that a protein required for photosystem II activity becomes functionally depleted during photoinhibition because protein synthesis or assembly into the membranes cannot keep up with the rate of its inactivation at excessively high PFDs. We also conclude that photoinhibition and light-dependent chilling sensitivity are manifestations of the same phenomenon.

CRYOPRESERVATION OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)

Cryopreserved Chlamydomonas reinhardtii cultures remained viable when frozen by cooling slowly to −55°C, then plunging into liquid nitrogen for at least 1 day of storage. High viability (>40%) was retained when cultures contained 2–10% (v/v) methanol as a cryoprotective agent prior to freezing, while dimethyl sulphoxide was ineffective. However, methanol was lethal to cells in the presence of light. Frozen cultures became non-viable within 24 h when stored at −80°C, whereas those stored below −130°C remained viable for at least several months. Highest viability was attained in cultures that were frozen and stored at a low cell density. High viability also required that frozen cultures be warmed rapidly and the cryoprotective agent removed immediately thereafter in preparation for culturing in liquid or on solid medium. Individual cell viability was determined by measurements of colony counts after cell plating and by the penetration of Evans blue dye into non-viable cells. Viability in bulk culture was co...

The effect of Ca2+ on oxygen evolution in membrane preparations from Anacystis nidulans

Divalent cations are known to be important for proper structure and function of photosynthetic membranes. In most cases Mg2+ appears to be the physiological ion of importance although ATPase activity in certain photosynthetic coupling factor preparations is Cap-dependent [I]. A direct role for Ca2+ in electron transport activities of isolated membranes from Cyanobacteria has been indicated [2,3]. Although Anacystis nidulans has been extensively used in studies of photosynthesis and other physiological problems, few investigations have utilized isolated photosynthetic membranes because of the difficulty in obtaining preparations which have retained their photochemical activities. Highly active preparations usually require incubation of cells with lysozyme under carefully controlled conditions, followed by separation of the membrane fraction from soluble components [4,5]. These preparations are often lengthy, and in our hands lead to variable activities from one preparation to the next. We report here a method for the rapid preparation of membrane fragments from Anacystis which give reproducibly high electron-transport activities when Ca2’ is present during cell breakage and during assays.

Separation of triacylglycerols and free fatty acids in microalgal lipids by solid-phase extraction for separate fatty acid profiling analysis by gas chromatography

Microalgal lipids were separated into two fractions, triacylglycerols (TAGs) and free fatty acids (FFAs), by solid-phase extraction employing sodium carbonate as the sorbent and dichloromethane (20% by volume) in n-hexane as the extracting solvent. The TAG fraction was then saponified, followed by acidification, extraction and tert-butyldimethylsilyl esterification. The FFA fraction was directly acidified, extracted and derivatized. From the lipid extracts of eight microalgal species examined, a total of 13 fatty acids were detected in the TAG fractions and nine were found in the FFA fractions, with at much higher total TAG content in all microalgae. Oleic acid was the most prominent fatty acid in three species, alpha-linolenic acid was more abundant in two others, and palmitic acid was present in highest concentration in the remaining three species.