A single species, Micromonas pusilla (Prasinophyceae), dominates the eukaryotic picoplankton in the Western English Channel

The class Prasinophyceae (Chlorophyta) contains several photosynthetic picoeukaryotic species described from cultured isolates. The ecology of these organisms and their contributions to the picoeukaryotic community in aquatic ecosystems have received little consideration. We have designed and tested eight new 18S ribosomal DNA oligonucleotide probes specific for different Prasinophyceae clades, genera, and species. Using fluorescent in situ hybridization associated with tyramide signal amplification, these probes, along with more general probes, have been applied to samples from a marine coastal site off Roscoff (France) collected every 2 weeks between July 2000 and September 2001. The abundance of eukaryotic picoplankton remained high (>10(3) cells ml(-1)) during the sampling period, with maxima in summer (up to 2 x 10(4) cells ml(-1)), and a single green algal species, Micromonas pusilla (Prasinophyceae), dominated the community all year round. Members of the order Prasinococcales and the species Bathycoccus prasinos (Mamiellales) displayed sporadic occurrences, while the abundances of all other Prasinophyceae groups targeted remained negligible.     

Six newly sequenced chloroplast genomes from prasinophyte green algae provide insights into the relationships among prasinophyte lineages and the diversity of streamlined genome architecture in picoplanktonic species

Background

Because they represent the earliest divergences of the Chlorophyta, the morphologically diverse unicellular green algae making up the prasinophytes hold the key to understanding the nature of the first viridiplants and the evolutionary patterns that accompanied the radiation of chlorophytes. Nuclear-encoded 18S rDNA phylogenies unveiled nine prasinophyte clades (clades I through IX) but their branching order is still uncertain. We present here the newly sequenced chloroplast genomes of Nephroselmis astigmatica (clade III) and of five picoplanktonic species from clade VI (Prasinococcus sp. CCMP 1194, Prasinophyceae sp. MBIC 106222 and Prasinoderma coloniale) and clade VII (Picocystis salinarum and Prasinophyceae sp. CCMP 1205). These chloroplast DNAs (cpDNAs) were compared with those of the six previously sampled prasinophytes (clades I, II, III and V) in order to gain information both on the relationships among prasinophyte lineages and on chloroplast genome evolution.

Results

Varying from 64.3 to 85.6 kb in size and encoding 100 to 115 conserved genes, the cpDNAs of the newly investigated picoplanktonic species are substantially smaller than those observed for larger-size prasinophytes, are economically packed and contain a reduced gene content. Although the Nephroselmis and Picocystis cpDNAs feature a large inverted repeat encoding the rRNA operon, gene partitioning among the single copy regions is remarkably different. Unexpectedly, we found that all three species from clade VI (Prasinococcales) harbor chloroplast genes not previously documented for chlorophytes (ndhJ, rbcR, rpl21, rps15, rps16 and ycf66) and that Picocystis contains a trans-spliced group II intron. The phylogenies inferred from cpDNA-encoded proteins are essentially congruent with 18S rDNA trees, resolving with robust support all six examined prasinophyte lineages, with the exception of the Pycnococcaceae.

Conclusions

Our results underscore the high variability in genome architecture among prasinophyte lineages, highlighting the strong pressure to maintain a small and compact chloroplast genome in picoplanktonic species. The unique set of six chloroplast genes found in the Prasinococcales supports the ancestral status of this lineage within the prasinophytes. The widely diverging traits uncovered for the clade-VII members (Picocystis and Prasinophyceae sp. CCMP 1205) are consistent with their resolution as separate lineages in the chloroplast phylogeny.     

Phylogenetic analyses of 18s rdna sequences reveal a new coccoid lineage of the prasinophyceae (Chlorophyta)

Phylogenetic analyses of 18S rDNA sequences from 25 prasinophytes, including 10 coccoid isolates, reveals that coccoid organisms are found in at least three prasinophyte lineages. The coccoid Ostreococcus tauri is included in the Mamiellales lineage and P ycnococcus provasolii is allied with the flagellate P seudoscourfieldia marina. A previously undescribed prasinophyte lineage is comprised of the coccoid Prasinococcus cf. capsulatus (CCMP 1407) and other isolates tentatively identified as Prasinococcus sp. (CCMP 1202, CCMP 1614, and CCMP 1194), as well as three unnamed coccoids (CCMP 1193, CCMP 1413, and CCMP 1220). No flagellate organisms are known from this lineage. Organisms of this new lineage share some characteristics of both the Pycnococcaceae and the Mamiellales, although relationships among these separate lineages were not supported by bootstrap analyses. An additional unnamed coccoid isolate (CCMP 1205) is separate from all major prasinophyte lineages. The analyses did not resolve the relationships among the major prasinophyte lineages, although they support previous conclusions that the Prasinophyceae are not monophyletic.     

Prasinoxanthin-constaining Prasinophyceae Discovered in Jiaozhou Bay, China

The class Prasinophyceae （Chlorophyta） contains some photosynthetic eukaryotic ultraplankton species characterized by containing prasinoxanthin. The existence and abundance of these organisms can be estimated by the diagnostic pigment. We detected the unique pigments of prasinoxanthin-containing Prasinophyceae in Jiaozhou Bay, China using high performance liquid chromatography （HPLC）. This was the first finding of this kind in Chinese seas. Using the ratio of prasinoxanthin to chlorophyll a, the abundance of prasinoxanthin-containing Prasinophyceae has been calculated. The average contribution of prasinoxanthin-containing Prasinophyceae to the chlorophyll a pool was 8.5% and 17.0% in May and August 2004 in Jiaozhou Bay, and the maximums were 25.9% and 36.3%. Size fractionated pigment analysis suggested that more than 80% of prasinoxanthin were in the fraction of 2-20 μm. According to the results of pigment and morphological analysis, the possible genera of prasinoxanthin-containing Prasinophyceae and the reasons for causing this high abundant phytoplankton in Jiaozhou Bay were discussed. This kind of phytoplankton can not be discovered in traditional biological investigation, but its contribution to the coastal ecosystem is significant enough to be studied further.     

Development of a 16S rRNA gene-based prototype microarray for the detection of selected actinomycetes genera

Actinomycetes are known for their secondary metabolites, which have been successfully used as drugs in human and veterinary medicines. However, information on the distribution of this group of Gram-positive bacteria in diverse ecosystems and a comprehension of their activities in ecosystem processes are still scarce. We have developed a 16S rRNA-based taxonomic microarray that targets key actinomycetes at the genus level. In total, 113 actinomycete 16S rRNA probes, corresponding to 55 of the 202 described genera, were designed. The microarray accuracy was evaluated by comparing signal intensities with probe/target-weighted mismatch values and the Gibbs energy of the probe/target duplex formation by hybridizing 17 non-actinomycete and 29 actinomycete strains/clones with the probe set. The validation proved that the probe set was specific, with only 1.3% of false results. The incomplete coverage of actinomycetes by a genus-specific probe was caused by the limited number of 16S rRNA gene sequences in databases or insufficient 16S rRNA gene polymorphism. The microarray enabled discrimination between actinomycete communities from three forest soil samples collected at one site. Cloning and sequencing of 16S rRNA genes from one of the soil samples confirmed the microarray results. We propose that this newly constructed microarray will be a valuable tool for genus-level comparisons of actinomycete communities in various ecological conditions.     

Feasibility of transferring fluorescent in situ hybridization probes to an 18S rRNA gene phylochip and mapping of signal intensities

DNA microarray technology offers the possibility to analyze microbial communities without cultivation, thus benefiting biodiversity studies. We developed a DNA phylochip to assess phytoplankton diversity and transferred 18S rRNA probes from dot blot or fluorescent in situ hybridization (FISH) analyses to a microarray format. Similar studies with 16S rRNA probes have been done determined that in order to achieve a signal on the microarray, the 16S rRNA molecule had to be fragmented, or PCR amplicons had to be <150 bp in length to minimize the formation of a secondary structure in the molecule so that the probe could bind to the target site. We found different results with the 18S rRNA molecule. Four out of 12 FISH probes exhibited false-negative signals on the microarray; eight exhibited strong but variable signals using full-length 18S RNA molecules. A systematic investigation of the probe's accessibility to the 18S rRNA gene was made using Prymenisum parvum as the target. Fourteen additional probes identical to this target covered the regions not tested with existing FISH probes. Probes with a binding site in the first 900 bp of the gene generated positive signals. Six out of nine probes binding in the last 900 bp of the gene produced no signal. Our results suggest that although secondary structure affected probe binding, the effect is not the same for the 18S rRNA gene and the 16S rRNA gene. For the 16S rRNA gene, the secondary structure is stronger in the first half of the molecule, whereas in the 18S rRNA gene, the last half of the molecule is critical. Probe-binding sites within 18S rRNA gene molecules are important for the probe design for DNA phylochips because signal intensity appears to be correlated with the secondary structure at the binding site in this molecule. If probes are designed from the first half of the 18S rRNA molecule, then full-length 18S rRNA molecules can be used in the hybridization on the chip, avoiding the fragmentation and the necessity for the short PCR amplicons that are associated with using the 16S rRNA molecule. Thus, the 18S rRNA molecule is a more attractive molecule for use in environmental studies where some level of quantification is desired. Target size was a minor problem, whereas for 16S rRNA molecules target size rather than probe site was important.     

Development and optimization of an 18S rRNA‐based oligonucleotide microarray for the fungal order Eurotiales

Aims: For identification of members of the fungal order Eurotiales, an 18S rRNA gene‐based oligonucleotide microarray was developed and optimized. Methods and Results: Eurotiales‐specific probes covering most members of the Eurotiales as well as species‐specific probes were designed and evaluated with three pure cultures (two fungi from the Eurotiales and one fungus from the Hypocreales). Nearly complete 18S rRNA genes of each reference culture were amplified and fluorescently labelled by random priming. Conclusions: Positive and negative hybridization results confirmed that the Eurotiales probes tested in this study could correctly identify members of the Eurotiales. The species‐specific probes were also capable of species‐level detection. Significance and Impact of the Study: These findings demonstrate the potential applications of a phylogenetic oligonucleotide microarray approach to characterizing fungal species and populations in environmental and other samples.     

Integrated Microbial Survey Analysis of Prokaryotic Communities for the PhyloChip Microarray

PhyloTrac is an integrated desktop application for analysis of PhyloChip microarray data. PhyloTrac combined with PhyloChip provides turnkey and comprehensive identification and analysis of bacterial and archaeal communities in complex environmental samples. PhyloTrac is free for noncommercial organizations and is available for all major operating systems at http://www.phylotrac.org/.The PhyloChip is a low-cost Affymetrix GeneChip microarray, developed at Lawrence Berkeley National Laboratory (LBNL), designed to detect and quantify abundance of bacterial and archaeal taxa using signature probes targeting all known 16S rRNA gene sequences. The second generation of the PhyloChip microarray targets nearly 9,000 operational taxonomic units (OTUs), with an average of 24 probes, each 25 bp long, and the upcoming third-generation PhyloChip application will target an even larger number of OTUs. Multiple, complex environments have been successfully analyzed using the PhyloChip microarray, including, among others, air (2), soil (1), the human lung (6), and the gut (9). PhyloChip microarrays are manufactured by Affymetrix, but to date, analysis has been available only from within LBNL, limiting the accessibility of the technology. PhyloTrac addresses this limitation by providing a standardized analysis package for the PhyloChip microarray, including microarray normalization, OTU quantification, multiple interactive visualizations, and integrated analytics.     

Differentiation of two very similar glaucomid ciliate morphospecies (Ciliophora, Tetrahymenida) by fluorescence in situ hybridization with 18S rRNA targeted oligonucleotide probes

Conventional, morphological identification of ciliates and other protozoa needs considerable experience and often is difficult as various staining methods must be applied. New molecular techniques, such as fluorescence in situ hybridization (FISH) with gene probes, are powerful means to overcome this problem. As a test case, the morphology of two very similar, and thus difficult to differentiate ciliate morphospecies, Glaucoma scintillans and Glaucomides bromelicola, were compared. They were then distinguished by applying the Ciliate-FISH technique with a set of eight 18S rRNA targeted oligonucleotide probes, four of which have been developed for specific detection of G. scintillans. The remaining four probes were designed to detect G. bromelicola in order to prove probe specificities by binding to the homologous target region of the probes mentioned before. The tests resulted in a clear and easy differentiation of the two species by fluorescence signals of three of the four tested probe pairs. Thus, FISH techniques are very useful for the identification and detection of protozoa and might be of great help studying geographical distributions of known taxa.     

Oligonucleotide microarray for 16S rRNA gene-based detection of all recognized lineages of sulfate-reducing prokaryotes in the environment

For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema- and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB).     

An oligonucleotide prokaryotic acidophile microarray: its validation and its use to monitor seasonal variations in extreme acidic environments with total environmental RNA

An oligonucleotide microarray that monitors prokaryotic diversity in extremely acidic environments has been developed. The oligonucleotide probes target most known acidophilic microorganisms, including members of the Nitrospira phylum, Acidithiobacillus genus, acidobacteria, sulfur reducing bacteria, Actinobacteria and Archaea of the Ferroplasma and Thermoplasma genera. The probes were tested for their specificity against the corresponding type strain by microarray hybridization using PCR-amplified fluorescent DNA of the 16S rRNA genes. The microarray was tested and validated against well-established molecular ecology techniques such as molecular cloning and sequencing and FISH by using samples obtained from a natural extremely acidic environment, the Río Tinto (SW Spain). Also, fluorescent labelled total environmental RNA from Río Tinto samples were used as targets for microarray hybridizations. This approach allowed the detection of the most metabolically active prokaryotes of the ecosystem by simultaneously checking probes against 16S and 23S rRNAs as well as other functional genes. Seasonal and spatial variations in the relative expression of specific rRNA genes have been detected between two sampling sites that differ in several physicochemical parameters, mainly iron and sulfur content.     

基因芯片技术检测3种肠道病原微生物方法的建立

目的:建立一种运用多重PCR和基因芯片技术检测和鉴定伤寒沙门氏菌、痢疾杆菌和单核细胞增生利斯特菌的方法。方法:分别选取伤寒沙门氏菌染色体ViaB区域中编码调控Vi抗原表达的基因(vipR)、痢疾杆菌编码侵袭质粒抗原H基因(ipaH)和单核细胞增生利斯特菌溶血素基因(hlyA)设计引物和探针,探针3'端进行氨基修饰,下游引物标记荧光素Cy3。在优化的PCR和杂交反应条件下,进行三重PCR扩增,产物与包括3种致病菌特异性探针的基因芯片杂交。在评价基因芯片的特异性和灵敏度之后,对临床样本进行检测。结果:只有3种目的致病菌的PCR产物在相应探针位置出现特异性信号,其他阴性细菌均无信号出现;3种致病菌的检测灵敏度均可达到103CFU/mL;检测30例临床样本的结果与常规细菌学培养结果一致。结论:所建立的可同时检测伤寒沙门氏菌、痢疾杆菌和单核细胞增生利斯特菌的基因芯片方法快速、准确,特异性高,重复性好,为3种肠道致病菌的快速检测和鉴定提供了新方法和新思路。     

Actin Phylogeny Identifies Mesostigma viride as a Flagellate Ancestor of the Land Plants

Green algae and land plants trace their evolutionary history to a unique common ancestor. This ``green lineage' is phylogenetically subdivided into two distinct assemblages, the Chlorophyta and the Streptophyta. The Chlorophyta includes the Chlorophyceae, Trebouxiophyceae, Ulvophyceae, and Prasinopohyceae, whereas the Streptophyta includes the Charophyceae plus the bryophytes, ferns, and all other multicellular land plants (Embryophyta). The Prasinophyceae is believed to contain the earliest divergences within the green lineage. Phylogenetic analyses using rDNA sequences identify the prasinophytes as a paraphyletic taxon that diverges at the base of the Chlorophyta. rDNA analyses, however, provide ambiguous results regarding the identity of the flagellate ancestor of the Streptophyta. We have sequenced the actin-encoding cDNAs from Scherffelia dubia (Prasinophyceae), Coleochaete scutata, Spirogyra sp. (Charophyceae), and the single-copy actin gene from Mesostigma viride (Prasinophyceae). Phylogenetic analyses show Mesostigma to be the earliest divergence within the Streptophyta and provide direct evidence for a scaly, biflagellate, unicellular ancestor for this lineage. This result is supported by the existence of two conserved actin-coding region introns (positions 20-3, 152-1), and one intron in the 5′-untranslated region of the actin gene shared by Mesostigma and the embryophytes. Received: 10 July 1997 / Accepted: 9 April 1998     

Quantitative assessment of picoeukaryotes in the natural environment by using taxon-specific oligonucleotide probes in association with tyramide signal amplification-fluorescence in situ hybridization and flow cytometry

Picoeukaryotes (cells of <3 micro m in diameter) contribute significantly to marine plankton biomass and productivity, and recently molecular studies have brought to light their wide diversity. Among the methods that have been used so far to quantify aquatic microorganisms, fluorescence in situ hybridization of oligonucleotide probes combined with flow cytometry offers the advantages of both high resolution for taxonomic identification and automated cell counting. However, cell losses, cell clumps, and low signal-to-background ratio have often been mentioned as major problems for routine application of this combination of techniques. We developed a new protocol associating tyramide signal amplification-fluorescence in situ hybridization and flow cytometry, which allows the detection of picoeukaryotes in cultures during both the exponential and stationary phases. The use of surfactant and sonication proved to be essential for the detection and quantification of picoeukaryotes from the natural environment, with as little as a few tenths of a milliliter of 3- micro m-pore-size prefiltered sea water. The routine application of the technique was tested along a coastal transect off Brittany (France), where the different groups of picoeukaryotes were investigated using already published specific probes and a newly designed probe that targets the order Mamiellales (Prasinophyceae, Chlorophyta). Among the picoeukaryotes, Mamiellales outnumbered by 1 order of magnitude both the cyanobacteria and the non-Chlorophyta, which were represented mainly by the Pelagophyceae class. Picoeukaryote abundance increased from open toward more estuarine water, probably following changes in water temperature and stability.     

Microplankton associations, biofacies and palaeoenvironment of the type lower Ludlow Series, Silurian

The acritarchs and prasinophyte algae from the type lower Ludlow Series of the Goggin Road section, Ludlow, England, are resolved into seven recurrent associations comprising taxa with similar environmental preferences. Endemic and environmentally sensitive associations of acritarchs and prasinophytes are identified and high-resolution fluctuations in the early Ludlow palaeoenvironment are established. An early Ludlow crisis in the acritarchs is recognized in the lower part of the Middle Elton Formation, when an abrupt palaeoenvironmental change in the Ludlow area resulted in a large decline in the abundance of the acritarchs, but allowed Tasmanites and retiolitid graptolites to flourish briefly. Cymbosphaeridium sp. A, Pulvinosphaeridium ludlowense and Multiplicisphaeridium arbusculum forma A are taxa possibly specialized, or produced as a response, to a stressed palaeoenvironment, as they are most abundant when other acritarchs and prasinophytes are uncommon. The low abundance of acritarchs and prasinophytes in the Upper Elton Formation may be related to high sedimentation rates and to the slumping of sediments caused by instability on the shelf of the Welsh Basin, or to lower plankton productivity.     

Assessment of cry1 gene contents of Bacillus thuringiensis strains by use of DNA microarrays

A single Bacillus thuringiensis strain can harbor numerous different insecticidal crystal protein (cry) genes from 46 known classes or primary ranks. The cry1 primary rank is the best known and contains the highest number of cry genes which currently totals over 130. We have designed an oligonucleotide-based DNA microarray (cryArray) to test the feasibility of using microarrays to identify the cry gene content of B. thuringiensis strains. Specific 50-mer oligonucleotide probes representing the cry1 primary and tertiary ranks were designed based on multiple cry gene sequence alignments. To minimize false-positive results, a consentaneous approach was adopted in which multiple probes against a specific gene must unanimously produce positive hybridization signals to confirm the presence of a particular gene. In order to validate the cryArray, several well-characterized B. thuringiensis strains including isolates from a Mexican strain collection were tested. With few exceptions, our probes performed in agreement with known or PCR-validated results. In one case, hybridization of primary- but not tertiary-ranked cry1I probes indicated the presence of a novel cry1I gene. Amplification and partial sequencing of the cry1I gene in strains IB360 and IB429 revealed the presence of a cry1Ia gene variant. Since a single microarray hybridization can replace hundreds of individual PCRs, DNA microarrays should become an excellent tool for the fast screening of new B. thuringiensis isolates presenting interesting insecticidal activity.     

16S rRNA gene-based phylogenetic microarray for simultaneous identification of members of the genus Burkholderia

For cultivation-independent and highly parallel analysis of members of the genus Burkholderia , an oligonucleotide microarray (phylochip) consisting of 131 hierarchically nested 16S rRNA gene-targeted oligonucleotide probes was developed. A novel primer pair was designed for selective amplification of a 1.3 kb 16S rRNA gene fragment of Burkholderia species prior to microarray analysis. The diagnostic performance of the microarray for identification and differentiation of Burkholderia species was tested with 44 reference strains of the genera Burkholderia , Pandoraea , Ralstonia and Limnobacter . Hybridization patterns based on presence/absence of probe signals were interpreted semi-automatically using the novel likelihood-based strategy of the web-tool PhyloDetect. Eighty-eight per cent of the reference strains were correctly identified at the species level. The evaluated microarray was applied to investigate shifts in the Burkholderia community structure in acidic forest soil upon addition of cadmium, a condition that selected for Burkholderia species. The microarray results were in agreement with those obtained from phylogenetic analysis of Burkholderia 16S rRNA gene sequences recovered from the same cadmium-contaminated soil, demonstrating the value of the Burkholderia phylochip for determinative and environmental studies.     

New insights into the nature and phylogeny of prasinophyte antenna proteins: Ostreococcus tauri, a case study

The basal position of the Mamiellales (Prasinophyceae) within the green lineage makes these unicellular organisms key to elucidating early stages in the evolution of chlorophyll a/b-binding light-harvesting complexes (LHCs). Here, we unveil the complete and unexpected diversity of Lhc proteins in Ostreococcus tauri, a member of the Mamiellales order, based on results from complete genome sequencing. Like Mantoniella squamata, O. tauri possesses a number of genes encoding an unusual prasinophyte-specific Lhc protein type herein designated "Lhcp". Biochemical characterization of the complexes revealed that these polypeptides, which bind chlorophylls a, b, and a chlorophyll c-like pigment (Mg-2,4-divinyl-phaeoporphyrin a5 monomethyl ester) as well as a number of unusual carotenoids, are likely predominant. They are retrieved to some extent in both reaction center I (RCI)- and RCII-enriched fractions, suggesting a possible association to both photosystems. However, in sharp contrast to previous reports on LHCs of M. squamata, O. tauri also possesses other LHC subpopulations, including LHCI proteins (encoded by five distinct Lhca genes) and the minor LHCII polypeptides, CP26 and CP29. Using an antibody against plant Lhca2, we unambiguously show that LHCI proteins are present not only in O. tauri, in which they are likely associated to RCI, but also in other Mamiellales, including M. squamata. With the exception of Lhcp genes, all the identified Lhc genes are present in single copy only. Overall, the discovery of LHCI proteins in these prasinophytes, combined with the lack of the major LHCII polypeptides found in higher plants or other green algae, supports the hypothesis that the latter proteins appeared subsequent to LHCI proteins. The major LHC of prasinophytes might have arisen prior to the LHCII of other chlorophyll a/b-containing organisms, possibly by divergence of a LHCI gene precursor. However, the discovery in O. tauri of CP26-like proteins, phylogenetically placed at the base of the major LHCII protein clades, yields new insight to the origin of these antenna proteins, which have evolved separately in higher plants and green algae. Its diverse but numerically limited suite of Lhc genes renders O. tauri an exceptional model system for future research on the evolution and function of LHC components.