Islandinium minutum subsp. barbatum subsp. nov. (Dinoflagellata), a New Organic-Walled Dinoflagellate Cyst from the Western Arctic: Morphology, Phylogenetic Position Based on SSU rDNA and LSU rDNA, and Distribution.

A study of modern sediment from the Western Arctic has revealed the presence of a distinctive brown-colored cyst with a spherical central body bearing unbranched processes that are usually solid with a small basal pericoel. Distinctive barbs project from some processes, and process tips are usually minutely expanded into conjoined barbs. The archeopyle is apical and saphopylic. This cyst corresponds to Islandinium? cezare morphotype 2 of Head et al. (2001, J. Quat. Sci., 16:621). Phylogenetic analyses based on the small and large subunit rRNA genes infer close relationship with Islandinium minutum, the type of which is that of the genus. Re-examination of specimens of I. minutum reveals the presence of minute barbs on its processes, but differences with Islandinium? cezare morphotype 2 remain based on size, process distribution, and barb development. Furthermore, the internal transcribed spacer shows I. minutum to be distinct from this morphotype. On the basis of these small but discrete differences, we propose the new subspecies Islandinium minutum subsp. barbatum subsp. nov. Molecular sequencing of other cysts encountered, namely Echinidinium karaense, an unidentified flattened cyst, and "Polykrikos quadratus", places them in the Monovela clade, the latter showing greater morphological variability than previously thought.

Structure Elucidation and in Vitro Toxicity of New Azaspiracids Isolated from the Marine Dinoflagellate Azadinium poporum.

Two strains of Azadinium poporum, one from the Korean West coast and the other from the North Sea, were mass cultured for isolation of new azaspiracids. Approximately 0.9 mg of pure AZA-36 (1) and 1.3 mg of pure AZA-37 (2) were isolated from the Korean (870 L) and North Sea (120 L) strains, respectively. The structures were determined to be 3-hydroxy-8-methyl-39-demethyl-azaspiracid-1 (1) and 3-hydroxy-7,8-dihydro-39-demethyl-azaspiracid-1 (2) by ¹H- and (13)C-NMR. Using the Jurkat T lymphocyte cell toxicity assay, (1) and (2) were found to be 6- and 3-fold less toxic than AZA-1, respectively.

Gymnodinium smaydae n. sp., a new planktonic phototrophic dinoflagellate from the coastal waters of Western Korea: morphology and molecular characterization.

The marine phototrophic dinoflagellate Gymnodinium smaydae n. sp. is described from cells prepared for light, scanning, and transmission electron microscopy. Also, sequences of the small (SSU) and large subunits (LSU) and the internal transcribed spacer region (ITS1-5.8S-ITS2) of ribosomal DNA were analyzed. This newly isolated dinoflagellate possessed nuclear chambers, nuclear fibrous connective, an apical groove running in a counterclockwise direction around the apex, and a major accessory pigment peridinin, which are four key features for the genus Gymnodinium. The epicone was conical with a round apex, while the hypocone was ellipsoid. Cells growing photosynthetically were 6.3-10.9 µm long and 5.1-10.0 µm wide, and therefore smaller than any other Gymnodinium species so far reported except Gymnodinium nanum. Cells were covered with polygonal amphiesmal vesicles arranged in 11 horizontal rows, and the vesicles were smaller than those of the other Gymnodinium species. This dinoflagellate had a sharp and elongated ventral ridge reaching half way down the hypocone, unlike other Gymnodinium species. Moreover, displacement of the cingulum was 0.4-0.6 × cell length while in other known Gymnodinium species it is less than 0.3 × cell length. In addition, the new species possessed a peduncle, permanent chloroplasts, pyrenoids, trichocysts, pusule systems, and small knobs along the apical furrow, but it lacked an eyespot, nematocysts, and body scales. The sequence of the SSU, ITS1-5.8S-ITS2, and LSU rDNA region differed by 1.5-3.8%, 6.0-17.4%, and 9.1-17.5%, respectively, from those of the most closely related species. The phylogenetic trees demonstrated that the new species belonged to the Gymnodinium clade at the base of a clade consisting of Gymnodinium acidotum, Gymnodinium dorsalisulcum, Gymnodinium eucyaneum, etc. Based on morphological and molecular data, we suggest that the taxon represents a new species, Gymnodinium smaydae n. sp.

First report of the photosynthetic dinoflagellate genus Azadinium in the Pacific Ocean: morphology and molecular characterization of Azadinium cf. poporum.

A strain of a dinoflagellate belonging to the genus Azadinium was obtained by the incubation of sediments collected from Shiwha Bay, Korea. This report of the genus Azadinium is the first outside of northern Europe and furthermore from the Pacific Ocean. The diagnostic morphological features of the isolate very closely resemble the recently described species Azadinium poporum isolated from the North Sea. However, the shape of the 3' apical plate and the occasional morphological variations unreported from A. poporum bring minor distinctions between strains from different locations. The DNA sequences of small subunit, ITS, and large subunit (LSU) rDNA differed by 0.2%, 2.6%, and 3.6%, respectively, from those of A. poporum, whereas the COI gene was identical to those found in all strains of Azadinium. Phylogenetic analyses of the ribosomal DNA regions generally positioned the Korean strain as a sister taxon of A. poporum. However, the Korean isolate tends to occupy a basal position within Azadinium species with ITS rDNA and LSU rDNA. Using liquid chromatography coupled with tandem mass spectrometry, no known azaspiracids were detected. The slight but discernible morphological differences, the distinct rDNA sequences, and the tendency of the Korean strain to diverge phylogenetically based on ITS rDNA and LSU rDNA from A. poporum do not enable us to clearly assign the isolate to A. poporum. However, these characteristics do not allow us to classify it as a distinct species, and it is therefore designated as Azadinium cf. poporum. The examination of more strains to find more diagnostic characteristics might enable the attribution of this material to a well-defined taxonomic position.

Sigma factors in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, as in most bacterial species, the expression of genes is tightly controlled by a repertoire of transcriptional regulators, particularly the so-called sigma (sigma) factors. The basic understanding of these proteins in bacteria has initially been described in Escherichia coli where seven sigma factors are involved in core RNA polymerase interactions and promoter recognition. Now, 7 years have passed since the completion of the first genome sequence of the opportunistic pathogen P. aeruginosa. Information from the genome of P. aeruginosa PAO1 identified 550 transcriptional regulators and 24 putative sigma factors. Of the 24 sigma, 19 were of extracytoplasmic function (ECF). Here, basic knowledge of sigma and ECF proteins was reviewed with particular emphasis on their role in P. aeruginosa global gene regulation. Summarized data are obtained from in silico analysis of P. aeruginosasigma and ECF including rpoD (sigma(70)), RpoH (sigma(32)), RpoF (FliA or sigma(28)), RpoS (sigma(S) or sigma(38)), RpoN (NtrA, sigma(54) or sigma(N)), ECF including AlgU (RpoE or sigma(22)), PvdS, SigX and a collection of uncharacterized sigma ECF, some of which are implicated in iron transport. Coupled to systems biology, identification and functional genomics analysis of P. aeruginosasigma and ECF are expected to provide new means to prevent infection, new targets for antimicrobial therapy, as well as new insights into the infection process.

Effects of warming and eutrophication on coastal phytoplankton production.

Phytoplankton production in coastal waters influences seafood production and human health and can lead to harmful algal blooms. Water temperature and eutrophication are critical factors affecting phytoplankton production, although the combined effects of warming and nutrient changes on phytoplankton production in coastal waters are not well understood. To address this, phytoplankton production changes in natural waters were investigated using samples collected over eight months, and under 64 different initial conditions, established by combining four different water temperatures (i.e., ambient T, +2, +4, and + 6 °C), and two different nutrient conditions (i.e., non-enriched and enriched). Under the non-enriched conditions, the effect of warming on phytoplankton production was significantly positive in some months, significantly negative in others, or had no effect. However, under enriched conditions, warming affected phytoplankton production positively in all months except one, when the salinity was as low as 6.5. These results suggest that nutrient conditions can alter the effects of warming on phytoplankton production. Of several parameters, the ratio of initial nitrate concentration to chlorophyll a concentration [NCCA, µM (µg L-1)-1] was one of the most critical factors determining the directionality of the warming effects. In laboratory experiments, when NCCA in the ambient or nutrient-enriched waters was ≥1.2, warming increased or did not change phytoplankton production with one exception; however, when NCCA was <1.2, warming did not change or decreased production. In the time series data obtained from the coastal waters of four target countries, when NCCA was 1.5 or more, warming increased phytoplankton production, whereas when NCCA was lower than 1.5, warming lowered phytoplankton production, Thus, it is suggested that NCCA could be used as an index for predicting future phytoplankton production changes in coastal waters.

Proteomic, microarray, and signature-tagged mutagenesis analyses of anaerobic Pseudomonas aeruginosa at pH 6.5, likely representing chronic, late-stage cystic fibrosis airway conditions.

Patients suffering from cystic fibrosis (CF) commonly harbor the important pathogen Pseudomonas aeruginosa in their airways. During chronic late-stage CF, P. aeruginosa is known to grow under reduced oxygen tension and is even capable of respiring anaerobically within the thickened airway mucus, at a pH of approximately 6.5. Therefore, proteins involved in anaerobic metabolism represent potentially important targets for therapeutic intervention. In this study, the clinically relevant "anaerobiome" or "proteogenome" of P. aeruginosa was assessed. First, two different proteomic approaches were used to identify proteins differentially expressed under anaerobic versus aerobic conditions. Microarray studies were also performed, and in general, the anaerobic transcriptome was in agreement with the proteomic results. However, we found that a major portion of the most upregulated genes in the presence of NO(3)(-) and NO(2)(-) are those encoding Pf1 bacteriophage. With anaerobic NO(2)(-), the most downregulated genes are those involved postglycolytically and include many tricarboxylic acid cycle genes and those involved in the electron transport chain, especially those encoding the NADH dehydrogenase I complex. Finally, a signature-tagged mutagenesis library of P. aeruginosa was constructed to further screen genes required for both NO(3)(-) and NO(2)(-) respiration. In addition to genes anticipated to play important roles in the anaerobiome (anr, dnr, nar, nir, and nuo), the cysG and dksA genes were found to be required for both anaerobic NO(3)(-) and NO(2)(-) respiration. This study represents a major step in unraveling the molecular machinery involved in anaerobic NO(3)(-) and NO(2)(-) respiration and offers clues as to how we might disrupt such pathways in P. aeruginosa to limit the growth of this important CF pathogen when it is either limited or completely restricted in its oxygen supply.

Effects of light intensity, temperature, and salinity on the growth and ingestion rates of the red-tide mixotrophic dinoflagellate Paragymnodinium shiwhaense.

Among mixotrophic dinoflagellates, the maximum mixotrophic growth rate of the red-tide dinoflagellate Paragymnodinium shiwhaense is relatively high, whereas mortality due to predation is low. To investigate the effects of major environmental parameters on P. shiwhaense, growth and ingestion rates of one strain of P. shiwhaense on the algal prey species Amphidinium carterae (also a dinoflagellate) were determined under various light intensities (0-500 µE m-2s-1), water temperatures (5-30 °C), and salinities (5-40). Cells of P. shiwhaense did not grow well in darkness but grew well at light intensities ≥ 10 µE m-2s-1. There were no significant differences in either growth or ingestion rates of P. shiwhaense fed A. carterae at light intensities between 10 and 500 µE m-2s-1. Furthermore, P. shiwhaense did not grow at 5 °C or ≥ 28 °C. Its growth rates between 7 and 26 °C were significantly affected by temperature, and the optimal temperature for maximal growth was 25 °C. With increasing salinity from 5 to 20, the growth rate of P. shiwhaense fed A. carterae increased and became saturated at salinities between 20 and 40, while the ingestion rate at salinities between 10 and 40 did not significantly change. Thus, overall, the growth and ingestion rates of P. shiwhaense fed A. carterae were affected by temperature and salinity, but not by light intensity other than darkness. These findings provide a beginning basis for understanding the ecology of this potentially harmful algal species in marine coastal ecosystems.

Identification of novel pathogenicity genes by PCR signature-tagged mutagenesis and related technologies.

Microbial pathogens possess a repertoire of virulence determinants that make unique contributions to bacterial fitness during infection. In this chapter, we focus on the recent progress and adaptations of signature-tagged mutagenesis (STM) by PCR instead of hybridization. This is a PCR-based STM mutation-based screening method using a population of bacterial mutants for the simultaneous identification of multiple virulence genes in microbial pathogens by negative selection. Modifications of STM developed in our laboratory have been applied to Pseudomonas aeruginosa PAO1. Screening of a collection of 6912 STM mutants in the rat chronic lung model of infection identified 214 P. aeruginosa STM mutants defective in virulence. For further studies, and to illustrate better the strategies that need to be utilized, we present detailed analysis of nine selected STM mutants. The data obtained indicate that in vivo, defects in virulence give a wide variety of phenotypes: defects in known virulence factors have been found, thereby validating the method; defects have also been found in orthologs with predicted functions, and in some genes whose functions cannot be predicted from databases. A general strategy and a simple scenario is discussed using the nine STM mutants selected for further characterization. PCR-based STM represent a genomics-based method for in vivo high-throughput screening of new virulence factors.

Cyst-theca relationship of the arctic dinoflagellate cyst Islandinium minutum (Dinophyceae) and phylogenetic position based on SSU rDNA and LSU rDNA.

Round brown spiny cysts constitute a morphological group common in high latitude dinoflagellate cyst assemblages. The dinoflagellate cyst Islandinium minutum (Harland et Reid) Head, Harland et Matthiessen is the main paleoecological indicator of seasonal sea-ice cover in the Arctic. Despite the importance of this cyst in paleoceanographical studies, its biological affinity has so far been unknown. The biological affinity of the species I. minutum and its phylogenetic position based on the small subunit ribosomal RNA gene (SSU rDNA) and the large subunit ribosomal RNA gene (LSU rDNA) were established from cyst incubation experiments in controlled conditions, optical and scanning electron microscopy, and single-cell PCR. The thecal motile cell obtained was undescribed. Although the motile cell was similar to Archaeperidinium minutum (Kofoid) Jörgensen, the motile cell of I. minutum lacked a transitional plate in the cingular series, which is present in Archaeperidinium spp. Islandinium minutum and Archaeperidinium spp. were paraphyletic in all phylogenetic analyses. Furthermore, Protoperidinium tricingulatum, which also lacks a transitional plate, was closely related to I. minutum and transfered to the genus Islandinium. Based on available data, it is clear that Islandinium is distinct from Archaeperidinium. Therefore, we considered Islandinium Head, Harland et Matthiessen as a non-fossil genus and emend its description, as well as the species I. minutum. This is the first description of a cyst-theca relationship and the first study that reports molecular data based on SSU rDNA and LSU rDNA on a species assigned to the genus Islandinium.

New azaspiracids in Amphidomataceae (Dinophyceae).

Azaspiracids (AZAs) are a group of lipophilic polyether toxins implicated in incidents of shellfish poisoning in humans, particularly in northern Europe, which are produced by the small marine dinoflagellate Azadinium spinosum. Other related species/strains of the Amphidomataceae have not been proven to date to contain any of the known azaspiracids. Closer analyses of these species/strains by triple quadrupole mass spectrometry in the precursor and product ion mode now revealed four new compounds with high similarity to azaspiracids, all of them with a characteristic m/z 348 fragment but with absence of the m/z 362 fragment. These compounds were detected in three species/strains, i.e. in North Sea isolates of Azadinium poporum (molecular mass: 845.5 Da), in a Korean isolate which has been designated as A. cf. poporum (molecular mass: 857.5 Da) and in Amphidoma languida isolated from Bantry Bay, Ireland (molecular masses: 815.5 and 829.5 Da). Cell quotas of roughly 2-20 fg per cell were in the same range as found for AZA-1/-2 in A. spinosum. Structures for all compounds were proposed by interpretation of fragmentation patterns and high resolution mass measurements using Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS).

Cooperative action of multiple cis-acting elements is required for N-myc expression in branchial arches: specific contribution of GATA3.

The precise expression of the N-myc proto-oncogene is essential for normal mammalian development, whereas altered N-myc gene regulation is known to be a determinant factor in tumor formation. Using transgenic mouse embryos, we show that N-myc sequences from kb -8.7 to kb +7.2 are sufficient to reproduce the N-myc embryonic expression profile in developing branchial arches and limb buds. These sequences encompass several regulatory elements dispersed throughout the N-myc locus, including an upstream limb bud enhancer, a downstream somite enhancer, a branchial arch enhancer in the second intron, and a negative regulatory element in the first intron. N-myc expression in the limb buds is under the dominant control of the limb bud enhancer. The expression in the branchial arches necessitates the interplay of three regulatory domains. The branchial arch enhancer cooperates with the somite enhancer region to prevent an inhibitory activity contained in the first intron. The characterization of the branchial arch enhancer has revealed a specific role of the transcription factor GATA3 in the regulation of N-myc expression. Together, these data demonstrate that correct N-myc developmental expression is achieved via cooperation of multiple positive and negative regulatory elements.

Comparative signature-tagged mutagenesis identifies Pseudomonas factors conferring resistance to the pulmonary collectin SP-A.

The pulmonary collectin, surfactant protein A (SP-A), is a broad spectrum opsonin with microbicidal membrane permeabilization properties that plays a role in the innate immune response of the lung. However, the factors that govern SP-A's microbial specificity and the mechanisms by which it mediates membrane permeabilization and opsonization are not fully understood. In an effort to identify bacterial factors that confer susceptibility or resistance to SP-A, we used comparative signature-tagged mutagenesis to screen a library of 1,680 Pseudomonas aeruginosa mutants for evidence of differential pulmonary clearance in SP-A-sufficient (SP-A) and SP-A-deficient (SP-A) mice. Two SP-A-sensitive P. aeruginosa mutants harboring transposon insertions in genes required for salicylate biosynthesis (pch) and phosphoenolpyruvate-protein-phosphotransferase (ptsP) were recovered. The mutants were indistinguishable from the parental wild-type PA01 with regard to opsonization by SP-A, but they exhibited increased susceptibility to SP-A-mediated membrane permeabilization. These results suggest that bacterial gene functions that are required to maintain membrane integrity play crucial roles in resistance of P. aeruginosa to the permeabilizing effects of SP-A.

In vivo functional genomics of Pseudomonas aeruginosa for high-throughput screening of new virulence factors and antibacterial targets.

Pseudomonas aeruginosa is a model for studying opportunistic pathogens that are highly resistant to most classes of antibiotics and cause chronic pulmonary infections. We have developed and adapted a multiplex polymerase chain reaction-based signature-tagged mutagenesis (STM) for high-throughput screening of a collection of 7968 P. aeruginosa mutants in a rat model of chronic respiratory infection. After three rounds of screening, a total of 214 mutants, representing transposition events into 148 open reading frames, were shown to be attenuated in lung infection and were retained for further analysis. As proof of concept supporting this technology, we identified 11 insertions in typical virulence genes such as those coding for pili implicated in motility, attachment and swarming, alginate synthesis and its expression, a mucus transcription regulator, extracellular enzymes such as alkaline protease, esterase and amino peptidase, a rhamnosyl surfactant transferase and a lipopolysaccharide glycosyl transferase. Detailed analysis of the 148 STM mutants, including seven auxotrophs, revealed insertions in 21 of the 26 known gene classes used to characterize sequenced bacterial genomes. We noted that at least 46% of STM mutants identified had insertions in hypothetical proteins or proteins of unknown function and that approximately 40% of all STM mutants had insertions in surface proteins including the outer membrane, the periplasm and the inner membrane. Interestingly, 11 STM mutants attenuated for lung infection were also identified in microarray and transcriptome for quorum sensing and mucoidy production. The remaining 130 mutants were systematically analysed for their capability to express fully known virulence factors. In addition, testing the ability of these mutants to infect alternative model host Drosophila melanogaster revealed 36 STM mutants defective in protease, twitching motility, swimming and swarming. Finally, we identified many genes, the activity of which in respiratory infection was not fully appreciated.