RESUMO
The cell cycle of Prochlorococcus, a prokaryote that accounts for a sizable fraction of the photosynthetic biomass in the eastern equatorial Pacific, progressed in phase with the daily light cycle. DNA replication occurred in the afternoon and cell division occurred at night. Growth rates were maximal (about one doubling per day) at 30 meters and decreased toward the surface and the bottom of the ocean. Estimated Prochlorococcus production varied between 174 and 498 milligrams of carbon per square meter per day and accounted for 5 to 19 percent of total gross primary production at the equator. Because Prochlorococcus multiplies close to its maximum possible rate, it is probably not severely nutrient-limited in this region of the oceans.
RESUMO
The minute photosynthetic prokaryote Prochlorococcus, which was discovered about 10 years ago, has proven exceptional from several standpoints. Its tiny size (0.5 to 0.7 microm in diameter) makes it the smallest known photosynthetic organism. Its ubiquity within the 40 degrees S to 40 degrees N latitudinal band of oceans and its occurrence at high density from the surface down to depths of 200 m make it presumably the most abundant photosynthetic organism on Earth. Prochlorococcus typically divides once a day in the subsurface layer of oligotrophic areas, where it dominates the photosynthetic biomass. It also possesses a remarkable pigment complement which includes divinyl derivatives of chlorophyll a (Chl a) and Chl b, the so-called Chl a2 and Chl b2, and, in some strains, small amounts of a new type of phycoerythrin. Phylogenetically, Prochlorococcus has also proven fascinating. Recent studies suggest that it evolved from an ancestral cyanobacterium by reducing its cell and genome sizes and by recruiting a protein originally synthesized under conditions of iron depletion to build a reduced antenna system as a replacement for large phycobilisomes. Environmental constraints clearly played a predominant role in Prochlorococcus evolution. Its tiny size is an advantage for its adaptation to nutrient-deprived environments. Furthermore, genetically distinct ecotypes, with different antenna systems and ecophysiological characteristics, are present at depth and in surface waters. This vertical species variation has allowed Prochlorococcus to adapt to the natural light gradient occurring in the upper layer of oceans. The present review critically assesses the basic knowledge acquired about Prochlorococcus both in the ocean and in the laboratory.
Assuntos
Cianobactérias/fisiologia , Água do Mar/microbiologia , Animais , Cianobactérias/química , Cianobactérias/ultraestrutura , Variação Genética/genética , Microscopia Eletrônica , Fotossíntese , Ficobilissomas , Filogenia , Pigmentos Biológicos/fisiologiaRESUMO
Recent molecular investigations of marine samples taken from different environments, including tropical, temperate and polar areas, as well as deep thermal vents, have revealed an unexpectedly high diversity of protists, some of them forming deep-branching clades within important lineages, such as the alveolates and heterokonts. Using the same approach on coastal samples, we have identified a novel group of protist small subunit (SSU) rDNA sequences that do not correspond to any phylogenetic group previously identified. Comparison with other sequences obtained from cultures of heterotrophic protists showed that the environmental sequences grouped together with Telonema, a genus known since 1913 but of uncertain taxonomic affinity. Phylogenetic analyses using four genes (SSU, Hsp90, alpha-tubulin and beta-tubulin), and accounting for gamma- and covarion-distributed substitution rates, revealed Telonema as a distinct group of species branching off close to chromist lineages. Consistent with these gene trees, Telonema possesses ultrastructures revealing both the distinctness of the group and the evolutionary affinity to chromist groups. Altogether, the data suggest that Telonema constitutes a new eukaryotic phylum, here defined as Telonemia, possibly representing a key clade for the understanding of the early evolution of bikont protist groups, such as the proposed chromalveolate supergroup.
Assuntos
DNA Ribossômico/classificação , Células Eucarióticas/classificação , Filogenia , Criptófitas/classificação , DNA Ribossômico/análise , Células Eucarióticas/ultraestrutura , Evolução Molecular , França , Proteínas de Choque Térmico HSP90/classificação , Proteínas de Choque Térmico HSP90/genética , Microscopia Eletrônica de Varredura , Análise de Sequência de DNA , Tubulina (Proteína)/classificação , Tubulina (Proteína)/genéticaRESUMO
Two Atlantic (SARG and NATL1) strains and one Mediterranean (MED) strain of Prochlorococcus sp., a recently discovered marine, free-living prochlorophyte, were grown over a range of "white" irradiances (lg) and under low blue light to examine their photoacclimation capacity. All three strains contained divinyl (DV) chlorophylls (Chl) a and b, both distinguishable from "normal" Chls by their red-shifted blue absorption maximum, a Chl c-like pigment at low concentration, zeaxanthin, and [alpha]-carotene. The presence of two phaeophytin b peaks in acidified extracts from both Atlantic strains grown at high lg suggests that these strains also had a normal Chl b-like pigment. In these strains, the total Chl b to DV-Chl a molar ratio decreased from about 1 at 7.5 [mu]mol quanta m-2 s-1 to 0.4 to 0.5 at 133 [mu]mol quanta m-2 s-1. In contrast, the MED strain always had a low DV-Chl b to DV-Chl a molar ratio, ranging between 0.13 at low lg and 0.08 at high lg. The discrepancies between the Atlantic and MED strains could result from differences either in the number of light-harvesting complexes (LHC) II per photosystem II or in the Chl b-binding capacity of the apoproteins constituting LHC II. Photosynthesis was saturated at approximately 5 fg C(fg Chl)-1 h-1 or 6 fg C cell-1 h-1, and growth was saturated at approximately 0.45 d-1 for both MED and SARG strains at 18[deg]C, but saturating irradiances differed between strains. Atlantic strains exhibited increased light-saturated rates and quantum yield for carbon fixation under blue light.
RESUMO
Flow cytometry (FCM) was successfully used to enumerate viruses in seawater after staining with the nucleic acid-specific dye SYBR Green-I. The technique was first optimized by using the Phaeocystis lytic virus PpV-01. Then it was used to analyze natural samples from different oceanic locations. Virus samples were fixed with 0.5% glutaraldehyde and deep frozen for delayed analysis. The samples were then diluted in Tris-EDTA buffer and analyzed in the presence of SYBR Green-I. A duplicate sample was heated at 80 degreesC in the presence of detergent before analysis. Virus counts obtained by FCM were highly correlated to, although slightly higher than, those obtained by epifluorescence microscopy or by transmission electron microscopy (r = 0.937, n = 14, and r = 0.96, n = 8, respectively). Analysis of a depth profile from the Mediterranean Sea revealed that the abundance of viruses displayed the same vertical trend as that of planktonic cells. FCM permits us to distinguish between at least two and sometimes three virus populations in natural samples. Because of its speed and accuracy, FCM should prove very useful for studies of virus infection in cultures and should allow us to better understand the structure and dynamics of virus populations in natural waters.
RESUMO
Two new oceanic free-living heterotrophic Heterokonta species with picoplanktonic size (< 2 microm) are described. Symbiomonas scintillans Guillou et Chrétiennot-Dinet gen. et sp. nov. was isolated from samples collected both in the equatorial Pacific Ocean and the Mediterranean Sea. This new species possesses ultrastructural features of the bicosoecids, such as the absence of a helix in the flagellar transitional region (found in Cafeteria roenbergensis and in a few bicosoecids), and a flagellar root system very similar to that of C. roenbergensis, Acronema sippewissettensis, and Bicosoeca maris. This new species is characterized by a single flagellum with mastigonemes, the presence of endosymbiotic bacteria located close to the nucleus, the absence of a lorica and a R3 root composed of a 6+3+x microtubular structure. Phylogenetical analyses of nuclear-encoded SSU rDNA gene sequences indicate that this species is close to the bicosoecids C. roenbergensis and Siluania monomastiga. Picophagus flagellatus Guillou et Chrétiennot-Dinet gen. et sp. nov. was collected in the equatorial Pacific Ocean. Cells are naked and possess two flagella. This species is characterized by the lack of a transitional helix and lateral filaments on the flagellar tubular hairs, the absence of siliceous scales, two unequal flagella, R1 + R3 roots, and the absence of a rhizoplast. SSU rDNA analyses place this strain at the base of the Chrysophyceae/Synurophyceae lineages.
Assuntos
Eucariotos/isolamento & purificação , Plâncton/isolamento & purificação , Animais , DNA de Plantas/genética , DNA Ribossômico/genética , Eucariotos/classificação , Eucariotos/genética , Eucariotos/ultraestrutura , Biologia Marinha , Mar Mediterrâneo , Oceano Pacífico , Filogenia , Plâncton/classificação , Plâncton/genética , Plâncton/ultraestrutura , Especificidade da Espécie , SimbioseRESUMO
A simple method was developed to preserve marine phytoplankton populations so that delayed flow cytometric analyses could be performed. The method consisted of immediate fixation with 1% glutaraldehyde (final concentration) followed by storage in liquid nitrogen. The method was tested on individual algal species and on natural samples from both coastal and pelagic waters. In most cases, it caused little cell loss and preserved well both forward angle light scatter and chlorophyll fluorescence, but phycoerythrin fluorescence sometimes was significantly increased. The technique performed best for the small-sized picoplankton (below 2 microns) such as Synechococcus cyanobacteria or the newly discovered oceanic prochlorophytes. For larger-sized cells it had to be applied on a case by case basis as some fragile species, particularly dinoflagellates and cryptophytes, were poorly preserved.
Assuntos
Citometria de Fluxo , Fitoplâncton , Plâncton , Preservação Biológica/métodos , Clorofila , Eucariotos , Fluorescência , Congelamento , Glutaral , FicoeritrinaRESUMO
Novel blue light-excited fluorescent dyes for nucleic acids (YOYO-1, YO-PRO-1, and PicoGreen) were tested on cultures of Escherichia coli and of a variety of marine prokaryotes. Results of flow cytometric DNA analyses were compared with those obtained with the UV-excited dyes bis-benzimide Hoechst 33342 or 4', 6-diamidino-2-phenylindole (DAPI). YOYO-1, YO-PRO-1, and PicoGreen can be used only on aldehyde-fixed cells and need to be supplemented with cofactors such as potassium, citrate, or EDTA. They are highly sensitive to ionic strength. Consequently, seawater culture samples cannot be stained directly with these dyes and require at least a 10-fold dilution with distilled water to obtain reliable fluorescence signals. After treatment with RNase, coefficients of variation for the G1 peak of the DNA distributions of the different strains tested with YOYO-1 or PicoGreen indicated in general an improvement over Hoechst 33342 staining. These novel dyes can be used to enumerate prokaryotic cells by flow cytometry, as demonstrated with E. coli. However, their sensitivity to ionic strength makes them unsuitable for cell cycle analysis in natural samples.
Assuntos
Benzoxazóis , DNA Bacteriano/análise , Escherichia coli/genética , Citometria de Fluxo/métodos , Corantes Fluorescentes , Compostos de Quinolínio , Compostos Orgânicos , Células ProcarióticasRESUMO
The ontogeny of immunoglobulin (Ig) synthesis was followed at both cellular and serological levels in the Mexican axolotl (Ambystoma mexicanum) using polyclonal antibodies recognizing all Ig molecules and a set of monoclonal antibodies (Mabs) specific for the C mu and Cv heavy Ig chain isotypes and for the light chain constituents shared by IgM and IgY molecules. Clusters of IgM- and of IgY-synthesizing lymphocytes, often located in separate sites, are first present in spleen sections of 7-week-old 25 mm larvae, about one month after differentiation of the spleen anlage (stage 39-40). In 12-week-old 30-35 mm larvae, the relative proportion of IgM- and IgY-synthesizing cells in the spleen is the same as that in adult animals. However, a marked enhancement of the spleen B cell compartment occurs from 5 to 9 months when Ig-positive cells represent about 88% of the lymphocytes population compared to 60% in adults. No structures equivalent to B cell germinal centers were observed at any stage of the spleen differentiation and cells, although often clustered in small groups, remain dispersed in the entire organ. The relative proportions of IgM and IgY B cells throughout the spleen remain constant during development (about 1 IgY+ cell for 5-6 IgM+ cells) and IgM molecules are first detected in the serum of 2.5-month-old larvae. The enhancement of the serum IgM level correlates well with the absolute number of IgM+ cells in the growing spleen. IgY molecules cannot be detected in the serum before the 7th month but their level quickly increases to reach about 60% of the adult value at 10 months. Thyroxine-induced metamorphosis or hyperimmunization of 4- to 6-month-old larvae had no effect upon the temporal expression of the Ig classes in serum.
Assuntos
Ambystoma mexicanum/crescimento & desenvolvimento , Ambystoma/crescimento & desenvolvimento , Expressão Gênica , Imunoglobulinas/biossíntese , Fatores Etários , Ambystoma mexicanum/genética , Ambystoma mexicanum/imunologia , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Mucosa Gástrica/metabolismo , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Linfócitos/citologia , Linfócitos/imunologia , Mesonefro/metabolismo , Microscopia de Contraste de Fase , Baço/embriologia , Baço/imunologiaRESUMO
The effect of light and dark on growth, DNA replication and cell division of two marine phytoplankters Thalassiosira weissflogii (a diatom) and Hymenomonas carterae (a coccolithophorid) was investigated using flow cytometry. The two species displayed very differing behavior. When transferred from light to prolonged darkness, all coccolithophorid cells were arrested at the beginning of the G1 stage of the cell cycle. When shifted back into light, they resumed cycling at a rate slightly slower than prior to arrest. In contrast, diatom cells were arrested either in the G1 or G2 stage of the cell cycle in the dark. Upon re-exposure to light, cells which had been dark-arrested in G1 resumed cycling at the same rate as prior to arrest, while cells arrested in G2 cycled much more slowly. These results suggest that in both species, light control of cell cycle progression is effective only over a restricted part of the cell cycle, as has been hypothesized by Spudich & Sager (J cell biol 83 (1980) 136) [38] for Chlamydomonas. In the coccolithophorid there is a single light-dependent segment located at the beginning of G1, whereas the diatom appears to have two such segments, one in G1 and the other in G2, corresponding to two different light requiring processes.
Assuntos
Ciclo Celular , Luz , Fitoplâncton/citologia , Plâncton/citologia , DNA/biossíntese , Escuridão , Interfase , Cinética , Mitose , Fitoplâncton/metabolismoRESUMO
Cell cycle phase durations of cultures of Hymenomonas carterae Braarud and Fagerl, a coccolithophore, and Thalassiosira weissflogii Grun., a centric diatom, in temperature-, light- or nitrogen-limited balanced growth were determined using flow cytometry. Suboptimal temperature caused increases in the duration of all phases of the cell cycle (though not equally) in both species, and the increased generation time of nitrogen-limited cells of both species was due almost wholly to expansion of G(1) phase. In H. carterae light limitation caused only G(1) phase to expand, but in T. weissflogii both G(2) + M and G(1) were affected. These results are discussed in relation to cell division phasing patterns of these two species and to models of phytoplankton growth. Simultaneous measurements of protein and DNA on individual cells indicated that under all conditions, the protein content of cells in G(1) was a constant proportion of that of G(2) + M cells. Simultaneous measurements of RNA and protein on each cell indicated that the amounts of these two cell constituents were always tightly correlated. Under conditions of nitrogen limitation both protein and RNA per cell decreased to less than one-third of the levels found in nonlimited cells. This indicates, at least for nitrogen-replete cells, that neither protein nor RNA levels are likely to act as the trigger for cell cycle progression. Strict control by cell size is also unlikely since mean cell volume decreased as growth rates were limited by light and nitrogen supply, but increased with decreasing temperature.
RESUMO
For many years, a small but dedicated group of scientists have been using flow cytometry for the evaluation of marine microorganisms. One of these scientists now provides us with a detailed series of protocols in this area, spelling out the variations in method and instrument operation that are crucial to the successful extraction of quality flow data from marine organisms. In addition, the use of a number of less frequently employed fluorescent probes gives some insight into alternative staining procedures. As our collection of microbiologically oriented techniques increases, this knowledge database becomes invaluable.
Assuntos
Bactérias/isolamento & purificação , Separação Celular/métodos , Citometria de Fluxo/métodos , Fitoplâncton/isolamento & purificação , Vírus/isolamento & purificação , Microbiologia da Água , Fluorescência , Espalhamento de RadiaçãoRESUMO
The effect of phosphorus (P) and nitrogen (N) additions on the Synechococcus cell cycle was tested with natural populations from the Mediterranean Sea in summer. In the absence of stimulation, the Synechococcus cell cycle was synchronized to the light-dark cycle. DNA synthesis began around 1600, a maximum of S-phase cells was observed at around dusk (2100), and a maximum of G(inf2)-phase cells was observed at around 2400. Addition of P (as PO(inf4)(sup3-)) caused, in all cases, a decrease in the fraction of cells in G(inf2) at around 1800, no change at around 2400, and an increase at around 1200 the next day, while addition of N (as NO(inf3)(sup-)) had no effect. We hypothesize that P addition induced a shortening of the G(inf1) phase, resulting in cells entering and leaving the S and G(inf2) phases earlier. These data suggest very strongly that the Synechococcus cells were P limited rather than N limited during this period of the year. In most cases, additions as low as 20 nM P induced a cell cycle response. From dose-response curves, we established that the P concentration inducing a 50% change in the percentage of cells in G(inf2) was low, close to 10 nM, at the beginning of the sampling period (30 June) and increased to about 50 nM by the end (9 July), suggesting a decrease in the severity of P limitation. This study extends recent observations that oligotrophic systems may be P rather than N limited at certain times of the year.
RESUMO
The novel dye SYBR Green I binds specifically to nucleic acids and can be excited by blue light (488-nm wavelength). Cell concentrations of prokaryotes measured in marine samples with this dye on a low-cost compact flow cytometer are comparable to those obtained with the UV-excited stain Hoechst 33342 (bis-benzimide) on an expensive flow cytometer with a water-cooled laser. In contrast to TOTO-1 and TO-PRO-1, SYBR Green I has the advantage of clearly discriminating both heterotrophic bacteria and autotrophic Prochlorococcus cells, even in oligotrophic waters. As with TOTO-1 and TO-PRO-1, two groups of heterotrophic bacteria (B-I and B-II-like types) can be distinguished. Moreover, the resolution of DNA distribution obtained with SYBR Green I is similar to that obtained with Hoechst 33342 and permits the analysis of the cell cycle of photosynthetic prokaryotes over the whole water column.
RESUMO
Picoplankton--cells with a diameter of less than 3 microm--are the dominant contributors to both primary production and biomass in open oceanic regions. However, compared with the prokaryotes, the eukaryotic component of picoplankton is still poorly known. Recent discoveries of new eukaryotic algal taxa based on picoplankton cultures suggest the existence of many undiscovered taxa. Conventional approaches based on phenotypic criteria have limitations in depicting picoplankton composition due to their tiny size and lack of distinctive taxonomic characters. Here we analyse, using an approach that has been very successful for prokaryotes but has so far seldom been applied to eukaryotes, 35 full sequences of the small-subunit (18S) ribosomal RNA gene derived from a picoplanktonic assemblage collected at a depth of 75 m in the equatorial Pacific Ocean, and show that there is a high diversity of picoeukaryotes. Most of the sequences were previously unknown but could still be assigned to important marine phyla including prasinophytes, haptophytes, dinoflagellates, stramenopiles, choanoflagellates and acantharians. We also found a novel lineage, closely related to dinoflagellates and not previously described.
Assuntos
Variação Genética , Plâncton/genética , Animais , Clonagem Molecular , DNA Ribossômico/genética , Células Eucarióticas/classificação , Dados de Sequência Molecular , Oceano Pacífico , Filogenia , Fitoplâncton/classificação , Fitoplâncton/genética , Plâncton/classificação , RNA Ribossômico 18S/genéticaRESUMO
The effect of light on the synchronization of cell cycling was investigated in several strains of the oceanic photosynthetic prokaryote Prochlorococcus using flow cytometry. When exposed to a light-dark (L-D) cycle with an irradiance of 25 micromol of quanta x m(-2) x s(-1), the low-light-adapted strain SS 120 appeared to be better synchronized than the high-light-adapted strain PCC 9511. Submitting L-D-entrained populations to shifts (advances or delays) in the timing of the "light on" signal translated to corresponding shifts in the initiation of the S phase, suggesting that this signal is a key parameter for the synchronization of population cell cycles. Cultures that were shifted from an L-D cycle to continuous irradiance showed persistent diel oscillations of flow-cytometric signals (light scatter and chlorophyll fluorescence) but with significantly reduced amplitudes and a phase shift. Complete darkness arrested most of the cells in the G1 phase of the cell cycle, indicating that light is required to trigger the initiation of DNA replication and cell division. However, some cells also arrested in the S phase, suggesting that cell cycle controls in Prochlorococcus spp. are not as strict as in marine Synechococcus spp. Shifting Prochlorococcus cells from low to high irradiance translated quasi-instantaneously into an increase of cells in both the S and G2 phases of the cell cycle and then into faster growth, whereas the inverse shift induced rapid slowing of the population growth rate. These data suggest a close coupling between irradiance levels and cell cycling in Prochlorococcus spp.
Assuntos
Ciclo Celular/fisiologia , Cianobactérias/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Luz , Ritmo Circadiano , Meios de Cultura , Cianobactérias/citologia , Escuridão , Citometria de FluxoRESUMO
Global identification of differentially regulated genes in prokaryotes is constrained because the mRNA does not have a 3' polyadenylation extension; this precludes specific separation of mRNA from rRNA and tRNA and synthesis of cDNAs from the entire mRNA population. Knowledge of the entire genome sequence of Synechocystis sp. strain PCC 6803 has enabled us to develop a differential display procedure that takes advantage of a short palindromic sequence that is dispersed throughout the Synechocystis sp. strain PCC 6803 genome. This sequence, designated the HIP (highly iterated palindrome) element, occurs in approximately half of the Synechocystis sp. strain PCC 6803 genes but is absent in rRNA and tRNA genes. To determine the feasibility of exploiting the HIP element, alone or in combination with specific primer subsets, for analyzing differential gene expression, we used HIP-based primers to identify light intensity-regulated genes. Several gene fragments, including those encoding ribosomal proteins and phycobiliprotein subunits, were differentially amplified from RNA templates derived from cells grown in low light or exposed to high light for 3 h. One novel finding was that expression of certain genes of the pho regulon, which are under the control of environmental phosphate levels, were markedly elevated in high light. High-light activation of pho regulon genes correlated with elevated growth rates that occur when the cells are transferred from low to high light. These results suggest that in high light, the rate of growth of Synechocystis sp. strain PCC 6803 exceeds its capacity to assimilate phosphate, which, in turn, may trigger a phosphate starvation response and activation of the pho regulon.
Assuntos
Cianobactérias/genética , Perfilação da Expressão Gênica/métodos , Genes Bacterianos , Genes Reguladores , Genoma Bacteriano , Sequência de Bases , Clonagem Molecular , Primers do DNA , Regulação Bacteriana da Expressão Gênica , Óperon , RNA Bacteriano/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
The past 10 years or so have seen the combination of molecular and biochemical techniques within the confines of cytometry. The use of flow cytometry in microbiology is finally coming of age. This unit carefully defines the criteria for evaluation of DNA and RNA in phytoplankton. Of course not everyone works with phytoplankton, but the methods outlined are very appropriately representative for other organisms. In addition, the unit discusses the methods for evaluating cell cycle and discriminating specific taxa using fluorescent oligonucleotide probes targeted to 18S rRNA.
Assuntos
Separação Celular/métodos , DNA/análise , Citometria de Fluxo/métodos , Fitoplâncton/metabolismo , RNA/análise , Corantes Fluorescentes/química , Sondas de Oligonucleotídeos/análise , RNA Ribossômico 18S/análiseRESUMO
DNA sequence, copy number, expression and phylogenetic relevance of the psbA gene from the abundant marine prokaryote P. marinus CCMP 1375 was analyzed. The 7 amino acids near the C-terminus missing in higher plant and in Prochlorothrix hollandica D1 proteins are present in the derived amino acid sequence. P. marinus contains only a single psbA gene. Thus, this organism lacks the ability to adapt its photosystem II by replacement of one type of D1 by another, as several cyanobacteria do. Phylogenetic trees suggested the D1-1 iso-form from Synechococcus PCC 7942 as the next related D1 protein and place P. marinus separately from Prochlorothrix hollandica among the cyanobacteria.
Assuntos
Cianobactérias/genética , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Sequência de Aminoácidos , Sequência de Bases , Cianobactérias/classificação , Dados de Sequência Molecular , Complexo de Proteína do Fotossistema II , Homologia de Sequência de AminoácidosRESUMO
Because of their tiny size (0.2 to 2 microns), oceanic picophytoplanktonic cells (either cultured strains or natural communities) are difficult to identify, and some basic questions concerning their taxonomy, physiology, and ecology are still largely unanswered. The present study was designed to test the suitability of in situ hybridization with rRNA fluorescent probes detected by flow cytometry for the identification of small photosynthetic eukaryotes. Oligonucleotide probes targeted against regions of the 18S rRNAs of Chlorophyta lineage (CHLO probe) and of non-Chlorophyta (NCHLO probe) algal species were designed. The CHLO and NCHLO probes, which differed by a single nucleotide, allowed discrimination of chlorophyte from nonchlorophyte cultured strains. The sensitivity of each probe was dependent upon the size of the cells and upon their growth stage. The mean fluorescence was 8 to 80 times higher for specifically labeled than for nonspecifically labeled cells in exponential growth phase, but it decreased sharply in stationary phase. Such taxon-specific probes should increase the applicability of flow cytometry for the rapid identification of cultured pico- and nanoplanktonic strains, especially those that lack taxonomically useful morphological features.