Polypeptide sequence of the chlorophyll a/b/c-binding protein of the prasinophycean alga Mantoniella squamata

The primary structure of the Chla/b/c-binding protein from Mantoniella squamata is determined. This is the first report that protein sequencing reveals one modified amino acid resulting in a LHCP-specific TFA-cleavage site. The comparison of the sequence of Mantoniella with other Chla/b-and Chla/c-binding proteins shows that the modified amino acid is located in a region which is highly conserved in all these proteins. The alignment also reveals that the LHCP of Mantoniella is related to the Chla/b-binding proteins. Finally, possible Chl-binding regions are discussed.Abbreviations a.m.u. atomic mass unit - LHC light-harvesting complex - LHC II major LHC of Photosystem II - LHCP light-harvesting chlorophyll-binding protein - LSIMS liquid secondary ion mass spectrometry - TFA trifluoroacetic acid     

The xanthophyll cycle of Mantoniella squamata converts violaxanthin into antheraxanthin but not to zeaxanthin: consequences for the mechanism of enhanced non-photochemical energy dissipation

The prasinophycean alga Mantoniella squamata uses in vivo an incomplete violaxanthin cycle. Although the violaxanthin cycle in Mantoniella is capable of converting violaxanthin to zeaxanthin, in intact cells only antheraxanthin accumulates during periods of strong illumination. Antheraxanthin enhances non-photochemical quenching of chlorophyll fluorescence. Inhibition of antheraxanthin synthesis by the de-epoxidase inhibitor dithiothreitol abolishes increased thermal energy dissipation. Antheraxanthin-dependent non-photochemical quenching, like zeaxanthin-mediated non-photochemical quenching in higher plants, is uncoupler-sensitive. Mantoniella squamata cells cultivated at high light intensities contain higher amounts of violaxanthin than cells grown at low light. The increased violaxanthin-cycle pool size in high-light-grown Mantoniella cells is accompanied by higher de-epoxidation rates in the light and by a greater capacity to quench chlorophyll fluorescence non-photochemically. Antheraxanthin-dependent amplification of non-photochemical quenching is discussed in the light of recent models developed for zeaxanthin- and diatoxanthin-mediated enhanced heat dissipation. Received: 4 September 1997 / Accepted: 22 December 1997     

Purification and identification of chlorophyll c1 from the green alga Mantoniella squamata

The prasinophycean alga Mantoniella squamata contains besides chlorophyll a and b a third chlorophyll c-like pigment in its light-harvesting antenna. This third chlorophyll was purified by reverse phase and polyethylene chromatography in order to identify its chemical structure. The absorption and fluorescence spectra were measured not only from the doubly purified pigment, but also from its Mg-free derivates. The spectra were compared with those of authentic chlorophyll c and of Mg-2,4-desethyl-2,4-divinylpheoporphyrin a₅ monomethyl ester which was isolated from Rhodobacter capsulata. The results show that the pigment from Mantoniella agrees best with chlorophyll c₁. In order to clarify the spectral data, chlorophyll c₁ and c₂, the pigment from Mantoniella and Mg-2,4-desethyl-2,4-divinylpheoporphyrin a₅ monomethyl ester were resolved by polyethylene chromatography. The chromatographic analysis clearly shows that the pigment from Mantoniella comigrates with chlorophyll c₁ and not with the bacterial pigment or chlorophyll c₂. Mantoniella is the first organism which has been demonstrated to contain chlorophyll a, b and c.     

Morphological and genetic diversity of Beaufort Sea diatoms with high contributions from the Chaetoceros neogracilis species complex

Seventy‐five diatom strains isolated from the Beaufort Sea (Canadian Arctic) in the summer of 2009 were characterized by light and electron microscopy (SEM and TEM), as well as 18S and 28S rRNA gene sequencing. These strains group into 20 genotypes and 17 morphotypes and are affiliated with the genera Arcocellulus, Attheya, Chaetoceros, Cylindrotheca, Eucampia, Nitzschia, Porosira, Pseudo‐nitzschia, Shionodiscus, Thalassiosira, and Synedropsis. Most of the species have a distribution confined to the northern/polar area. Chaetoceros neogracilis and Chaetoceros gelidus were the most represented taxa. Strains of C. neogracilis were morphologically similar and shared identical 18S rRNA gene sequences, but belonged to four distinct genetic clades based on 28S rRNA, ITS‐1 and ITS‐2 phylogenies. Secondary structure prediction revealed that these four clades differ in hemi‐compensatory base changes (HCBCs) in paired positions of the ITS‐2, suggesting their inability to interbreed. Reproductively isolated C. neogracilis genotypes can thus co‐occur in summer phytoplankton communities in the Beaufort Sea. C. neogracilis generally occurred as single cells but also formed short colonies. It is phylogenetically distinct from an Antarctic species, erroneously identified in some previous studies as C. neogracilis, but named here as Chaetoceros sp. This work provides taxonomically validated sequences for 20 Arctic diatom taxa, which will facilitate future metabarcoding studies on phytoplankton in this region.     

Energy transfer and pigment composition in three chlorophyll b-containing light-harvesting complexes isolated from Mantoniella squamata (Prasinophyceae), Chlorella fusca (Chlorophyceae) and Sinapis alba

Light-harvesting Chl a/b protein complexes were isolated from the higher plant Sinapis alba, the green alga Chlorella fusca, and the prasinophycean alga Mantoniella squamata by mild gel electrophoresis. The energy transfer from chlorophyll b and the accessory xanthophyll was measured by means of fluoresence spectroscopy at 77 K. The pigment composition of the isolated antenna complexes was determined by high performance liquid chromatography in order to calculate the number of light absorbing molecules per chlorophyll a in the different light-harvesting complexes. These results were complemented by the quantitation of the pigments in total thylakoids as well as in the different electrophoretic fractions. On the basis of these data the in vivo ratios of xanthophylls per chlorophyll a could be estimated. The results show that the light-harvesting complexes from Chlorella and from Sinapis exhibit identical ratios of total xanthophylls per chlorophyll a. By contrast, in the prasinophycean alga Mantoniella, the light-harvesting complex markedly differs from the other chlorophyll b containing proteins. It contains, in addition to neoxanthin and violaxanthin, high amounts of prasinoxanthin and its epoxide, which contribute significantly to light absorption. The concentration of chlorophyll b in the complex is very much higher in the antenna of Mantoniella than in those of Chlorella and Sinapis. Furthermore, it must be emphasized that in addition to chlorophyll b, a third chlorophyll species acts in the energy transfer to chlorophyll a. This chlorophyll c-like pigment is found to be present in a concentration which improves very efficiently the absorption in blue light. In light of these results it can be concluded that the absorption cross section in Mantoniella is higher not only because of an enhanced number of light-harvesting particles in the membrane, but also because of a higher ratio of accessory pigments to chlorophyll a.Abbreviations Chl Chlorophyll - FP Free Pigments - HPLC High Performance Liquid Chromatography - LHC Light-harvesting Chlorophyll protein complex - PAGE Polyacrylamide Gel Electrophoresis - PS Photosystem     

Genomic diversity and CRISPR-Cas systems in the cyanobacterium Nostoc in the High Arctic

Nostoc (Nostocales, Cyanobacteria) has a global distribution in the Polar Regions. However, the genomic diversity of Nostoc is little known and there are no genomes available for polar Nostoc. Here we carried out the first genomic analysis of the Nostoc commune morphotype with a recent sample from the High Arctic and a herbarium specimen collected during the British Arctic Expedition (1875–76). Comparisons of the polar genomes with 26 present-day non-polar members of the Nostocales family highlighted that there are pronounced genetic variations among Nostoc strains and species. Osmoprotection and other stress genes were found in all Nostoc strains, but the two Arctic strains had markedly higher numbers of biosynthetic gene clusters for uncharacterised non-ribosomal peptide synthetases, suggesting a high diversity of secondary metabolites. Since viral–host interactions contribute to microbial diversity, we analysed the CRISPR-Cas systems in the Arctic and two temperate Nostoc species. There were a large number of unique repeat-spacer arrays in each genome, indicating diverse histories of viral attack. All Nostoc strains had a subtype I-D system, but the polar specimens also showed evidence of a subtype I-B system that has not been previously reported in cyanobacteria, suggesting diverse cyanobacteria–virus interactions in the Arctic.     

FLAGELLAR HAIRS IN PRASINOPHYTES (CHLOROPHYTA): ULTRASTRUCTURE AND DISTRIBUTION ON THE FLAGELLAR SURFACE1

The flagellar hair ultrastructure of 16 strains of species of the prasinophycean genera Mantoniella, Mamiella, Pseudoscourfieldia, Nephroselmis, Tetraselmis, Scherffelia, Pterosperma, and Pyraminonas was examined in detail by whole-mount electron microscopy. The flagellar hairs of all genera displayed a high degree of ultrastructural complexity that was completely conserved within each strain. In all strains, flagellar hairs occurred on the sides of the flagella (lateral hairs); in several strains, special flagellar hairs also were found on the flagellar tips (tip hairs; absent in the Chlorodendrales and in Nephroselmis). Two groups of lateral hairs were distinguished: 1) T-hairs (“Tetraselmis-type” flagellar hairs), characterized by a smooth, tubular shaft of ca. 15 nm diameter and an overall length of 0.5–1.3 μm, and 2) P_t-hairs (“Pterosperma-type lateral flagellar hairs”), which were considerably longer (ca. 1.5–5.4 μm), characterized by a thick shaft of ca. 30 nm diameter, which was covered with a layer of regularly spaced small particles of ca. 10 nm diameter. In both groups of flagellar hairs, a strain-specific number of subunits (1–101) in linear arrangement was attached to the distal end of the shaft. Tip hairs were either structurally related to T-hairs (Mamiellales, Pseudoscourfieldia) or represented a separate group, P_t-hairs (“Pterosperma-type flagellar tip hairs”; Pterosperma, Pyramimonas). In four genera (Mantoniella, Mamiella, Pseudoscourfieldia, Nephroselmis), both groups of lateral hairs occurred together on the same cell. Interestingly in these taxa the P_t-hairs were exclusively attached to the shorter immature flagella (no. 2), but, in contrast, in Mantoniella and Pseudoscourfieldia the tip hairs were restricted to the longer mature flagellum (no. 1). Thus, flagella of different developmental status differ in their hair-scale complement. The occurrence, distribution, and ultrastructure of flagellar hairs can be used to identify and classify prasinophytes at all taxonomic levels.     

Establishing a New Species Encephalitozoon pogonae for the Microsporidian Parasite of Inland Bearded Dragon Pogona vitticeps Ahl 1927 (Reptilia,Squamata, Agamidae)

The microsporidium parasitizing Inland Bearded Dragons Pogona vitticeps, and developing primarily in macrophages within foci of granulomatous inflammation of different organs, is described as a new species Encephalitozoon pogonae. Establishing the new species was based on sequencing the ITS‐SSUrDNA region of the ribosomal gene and consequent SSUrDNA‐inferred phylogenetic analyses, as well as on comparison of pathogenesis, host specificity, and ultrastructure among Encephalitozoon species and isolates. The new species is closely related to E. lacertae and E. cuniculi. Analysis of the literature suggests that this microsporidium has been reported previously as an unidentified microsporidian species or isolate of E. cuniculi and may represent a common infection in bearded dragons. All stages of E. pogonae develop in parasitophorous vacuoles. Uninucleate spores on methanol‐fixed smears measured 2.1 × 1.1 μm, range 1.7–2.6 × 0.9–1.7 μm; on ultrathin sections spores measured 0.8–1.1 × 1.8–2.2 μm. Ultrastructural study revealed 3–6 polar filament coils, a mushroom‐shaped polar disk, and a polar sac embracing half of the volume occupied by the lamellar polaroplast. In activated spores, polar filament everted eccentrically. The overall morphology and intracellular development of E. pogonae were similar to other Encepahalitozoon spp. We also review the existing data on microsporidia infecting reptiles.     

Comparative assessment of single and joint effects of diuron and Irgarol 1051 on Arctic and temperate microalgae using chlorophyll a fluorescence imaging

Ship groundings and ice-breakers can cause pollution of the polar environment with antifouling biocides such as diuron and Irgarol 1051. The present study used pulse amplitude modulated fluorometry to compare single and joint toxicities of diuron and Irgarol 1051 on two freshwater taxa of microalgae (Chlorella and Chlamydomonas) originating from Arctic and temperate regions. 30 min acute toxicity tests using chlorophyll a (Chl a) fluorescence revealed that Arctic strains of microalgae were more sensitive to herbicides than their temperate counterparts. Diuron and Irgarol 1051 had equal toxicities in the Arctic species, while Irgarol 1051 was more toxic (EC₅₀ = 5.55–14.70 μg L⁻¹) than diuron (EC₅₀ = 12.90–>40 μg L⁻¹) in the temperate species. Toxicity assessment of various mixtures of diuron and Irgarol 1051 revealed antagonistic, additive, and synergistic effects. Our data suggest that herbicides can adversely affect photosynthesis in Arctic microalgae at relatively low levels, and their impact can increase under complex mixture conditions.     

Molecular phylogeny of the spiny-surfaced species of the dinoflagellate Prorocentrum with the description of P. thermophilum sp. nov. and P. criophilum sp. nov. (Prorocentrales,Dinophyceae)

Spiny-surfaced species of Prorocentrum form harmful algal blooms, and its taxonomic identity is obscure due to the size and shape variability. Molecular phylogenies reveal two major clades: one for P. cordatum with sequences mainly retrieved as P. minimum, and the other for P. shikokuense with sequences also retrieved as P. dentatum and P. donghaiense. Several closely related clades still need to be characterized. Here, we provide nuclear SSU and LSU rRNA genes, and nuclear ITS region (ITS1-5.8S gene-ITS2) sequences of the strain CCMP3122 isolated from Florida (initially named P. donghaiense) and strains Prorocentrum sp. RCC6871–2 from the Ross Sea, Antarctica. We describe Prorocentrum thermophilum sp. nov. based on the strain CCMP3122, a species also distributed in the open waters of the Gulf of Mexico, New Zealand, and the Arabian Gulf; and Prorocentrum criophilum sp. nov. based on the strain RCC6872, which is distributed in the Antarctic Ocean and Arctic Sea. Prorocentrum thermophilum is roundish (~14 μm long, ~12 μm wide), with an inconspicuous anterior spine-like prolongation under light microscopy, valves with tiny, short knobs (5–7 per μm²), and several (<7) large trichocyst pores (~0.3 μm) in the right valve, as well as smaller pores (~0.15 μm). Prorocentrum criophilum is round in valve view (~11 μm long, 10 μm wide) and asymmetrically roundish in lateral view, the periflagellar area was not discernible under light microscopy, valves with very tiny, short knobs (6–10 per μm²), and at least 12 large pores in the right valve. Other potentially undescribed species of spiny-surfaced Prorocentrum are discussed.     

Rhinomonas nottbecki n. sp. (Cryptomonadales) and Molecular Phylogeny of the Family Pyrenomonadaceae

The cryptomonad Rhinomonas nottbecki n. sp., isolated from the Baltic Sea, is described from live and fixed cells studied by light, scanning, and transmission electron microscopy together with sequences of the partial nucleus‐ and nucleomorph‐encoded 18S rRNA genes as well as the nucleus‐encoded ITS1, 5.8S, ITS2, and the 5′‐end of the 28S rRNA gene regions. The sequence analyses include comparison with 43 strains from the family Pyrenomonadaceae. Rhinomonas nottbecki cells are dorsoventrally flattened, obloid in shape; 10.0–17.2 μm long, 5.5–8.1 μm thick, and 4.4–8.8 μm wide. The inner periplast has roughly hexagonal plates. Rhinomonas nottbecki cells resemble those of Rhinomonas reticulata, but the nucleomorph 18S rRNA gene of R. nottbecki differs by 2% from that of R. reticulata, while the ITS region by 11%. The intraspecific variability in the ITS region of R. nottbecki is 5%. In addition, the predicted ITS2 secondary structures are different in R. nottbecki and R. reticulata. The family Pyrenomonadaceae includes three clades: Clade A, Clade B, and Clade C. All Rhinomonas sequences branched within the Clade C, while the genus Rhodomonas is paraphyletic. The analyses suggest that the genus Storeatula is an alternating morphotype of the genera Rhinomonas and Rhodomonas and that the family Pyrenomonadaceae includes some species that were described multiple times, as well as novel species.     

Genotypic characterization of Brochothrix spp. isolated from meat,poultry and fish

Aim: To study genotypic diversity of isolates of Brochothrix thermosphacta recovered from meat, poultry and fish. Methods and Results: A total of 27 bacteria isolated from 19 samples of meat, poultry and fish were identified phenotypically and genotypically using PCR amplification of 16S‐23S rDNA intergenic transcribed spacer (ITS‐PCR), repetitive sequence‐based PCR (rep‐PCR) and 16S rDNA sequencing. Using ITS‐PCR, all bacteria showed the same DNA profile as the reference strains of Br. thermosphacta, allowing typing of the isolates at species level. Using 16S rDNA sequencing, all isolates were identified, at genus and species level, as Br. thermosphacta. Identification as Br. campestris was observed with a lower, but very close, level of similarity. Rep‐PCR was more discriminatory than ITS‐PCR and allowed differentiation of four subgroups among the isolates. Conclusion: Minor genotypic differences among Br. thermosphacta strains from meat, poultry and fish were observed. Significance and Impact of the Study: A rudimentary exploration of genotypic differences of Br. thermosphacta from meat, poultry and fish resulted in preliminary confirmation of the suitability of ITS‐PCR for typing Br. thermosphacta and confirmed the value of rep‐PCR fingerprinting to discriminate between Br. thermosphacta strains.     

Distribution of polar cod and age-0 fish in the U.S. Beaufort Sea

Little is known about species composition, distribution, and abundance of pelagic fish in the U.S. portion of the Beaufort Sea continental shelf and slope. To inventory the community and describe pelagic fish distributions relative to water characteristics, a systematic survey was conducted in August 2008. Acoustics (38 kHz), midwater trawling, and CTD casts were used to sample water depths from 20 to 500 m. Age-1+ polar cod (Boreogadus saida) was the dominant fish species, with peak densities of 155,000 # ha⁻¹ at bottom depths of 100–350 m. Age-0 fish (polar cod, sculpin (Cottidae family), and eelblenny (Lumpenus sp.)), dominated the pelagic biomass at bottom depths between 20 and 75 m, with peak densities of 160,000 # ha⁻¹, but were also found in surface waters at bottom depths >75 m. Age-1+ polar cod were associated with cold, saline waters likely of Chukchi Sea origin and mirrored published foraging distributions for beluga whales (Delphinapterus leucas). Conversely, age-0 fish were found in warm, fresher water, likely of ice melt and/or riverine origin, throughout the study area. This study provides a necessary baseline for the development of Arctic assessment surveys and management plans for polar cod.     

Phylogeographical pattern of Euplotes nobilii,a protist ciliate with a bipolar biogeographical distribution

Nuclear (18S and ITS) and mitochondrial (16S) ribosomal RNA gene sequences were determined from genetically distinct wild‐type strains of Antarctic (nine strains), Fuegian (four strains), Greenland (nine strains) and Svalbard (three strains) populations of the marine ciliate, Euplotes nobilii, and analysed for their nucleotide polymorphisms. A close genetic homogeneity was found within and between the Antarctic and Fuegian populations, while more significant levels of genetic differentiation were detected within and between the two Arctic populations, as well as between these populations and the Antarctic/Fuegian ones. The phylogeographical pattern that was derived from these data indicates that gene flow is not limited among Arctic populations; it equally connects the Arctic and Antarctic populations either directly, or through the Fuegian population. This indication reinforces previous evidence from laboratory assays of mating interactions between some of the strains analysed in this work that Southern and Northern polar populations of E. nobilii belong to a unique, panmictic population that substantially share the same gene pool.     

Examination of prezygotic and postzygotic isolating barriers in tropical Ulva (Ulvophyceae,Chlorophyta): evidence for ongoing speciation

Phylogenetic clades based on DNA sequences such as the chloroplast rbcL gene and the nuclear ITS region are frequently used to delimit algal species. However, these molecular markers cannot accurately delimit boundaries among some Ulva species. Although Ulva reticulata and Ulva ohnoi occasionally bloom in tropical to warm‐temperate regions and are clearly distinguishable by their reticulate or plain blade morphology, they have few or no sequence divergences in these molecular markers and form a monophyletic clade. In this study, to clarify the speciation and species delimitation in the U. reticulata‐ohnoi complex clade, reproductive relationships among several sexual strains from the Philippines and Japan including offspring that originated from the type specimen of U. ohnoi were examined by culturing and hybridization in addition to the ITS‐based analysis. As a result, both prezygotic and postzygotic reproductive isolation were revealed to occur between genetically perforated U. reticulata and imperforate U. ohnoi. They were also separated on the basis of sequence analysis of the ITS region. That strongly supports that the two taxa are independent biological species. Although no prezygotic barrier among the Philippine and Japanese strains of U. reticulata was observed, unexpectedly zoospores produced by hybrid sporophytes in some of their combinations mostly failed to develop, indicating partial formation of a postzygotic barrier despite a 0.2% divergence in the ITS sequence. These findings suggest speciation is still ongoing in U. reticulata.     

Deinococcus aestuarii sp. nov. and Deinococcus aquaedulcis sp. nov., two novel resistant bacteria isolated from pearl river estuary

Two novel species of the genus Deinococcus, designated SYSU M49105^T and SYSU M42101^T, were isolated from freshwater samples of the Pearl River estuary in Guangdong, China. Phylogenetic analysis using 16S rRNA gene sequence indicated that strains SYSU M49105^T and SYSU M42101^T showed the highest sequence similarities to Deinococcus aetherius JCM 11751 ^T (93.6%) and Deinococcus multiflagellatus NBRC 112888 ^T (97.3%), respectively. Cells of both strains were Gram-staining positive, aerobic, coccus-shaped, oxidase-negative and non-motile. The cell wall contained meso-diaminopimelic acid as their diagnostic diamino acid. MK-8 was the predominant respiratory quinone for both strains. The polar lipid profile of SYSU M49105^T contained two unidentified phosphoglycolipids, nine unidentified glycolipids, and five unidentified polar lipids. SYSU M42101^T had one unidentified phosphoglycolipid, nine unidentified glycolipids, one unidentified aminophospholipid and four unidentified polar lipids. The major fatty acids of strains SYSU M49105^T and SYSU M42101^T were summed feature 3 (C_16:1 ω7c and/ or C_16:1 ω6c) and C_16:0. The G?+?C contents of the novel isolates based on genomic DNAs were 69.6% and 67.4%, respectively. On the basis of phenotypic, genotypic and phylogenetic data, strains SYSU M49105^T and SYSU M42101^T should be considered to represent two novel species in the genus Deinococcus, for which the names Deinococcus aestuarii sp. nov. and Deinococcus aquaedulcis sp. nov. were proposed with the type strains SYSU M49105^T (=?KCTC 43258 ^T?=?CGMCC 1.18609 ^T) and SYSU M42101^T (=?KCTC 43257 ^T?=?CGMCC 1.18614 ^T), respectively.

     

The Existence of Chlorophyll c in the Chl b-Containing,Light-Harvesting Complex of the Green Alga Mantoniella squamata (Prasinophyceae)

The prasinophycean alga Mantoniella contains, in addition to Chl a and b, at least a third green pigment which is functionally active in the light-harvesting antenna. This third Chl was isolated in order to elucidate its chemical structure. The absorption and fluorescence spectra were measured not only from the purified pigment but also from its pheophytin and its methylpheophorbide. The spectra were compared with those of authentic Chl c-1 and c-2, which were isolated from the diatom Nitzschia sp. and with Mg-DVPP (purified from Rhodobacter). The results show that the pigment from Mantoniella compares best with Chl c-1. In order to clarify the spectral data, Chl c-1 and c-2, Mg-DVPP, and the pigment from Mantoniella were subjected to a chromatographic system that is able to separate these porphyrins. The chromatographic analysis clearly shows that the pigment from Mantoniella co-migrates with Chl c-1 and not with the bacterial pigment. Mantoniella is the first organism which has been demonstrated to contain Chl a, b, and authentic c.