Genotypic relationships between Prochloron samples from different localities and hosts as determined by DNA-DNA reassociations

Genotypic relationships between seven Prochloron samples isolated from different didemnid ascidian hosts collected at the Palau archipelago and Munda (Solomon Islands) and one cyanobacterial (Synechocystis) strain were determined by DNA-DNA reassociations. Thermal stability values of DNA-DNA hybrids indicate that all Prochloron samples involved are mutually very closely related and only slightly related with the Synechocystis strain. It is concluded that the Prochloron samples tested are representatives of one and the same species.     

Deoxyribonucleic acid base composition and genome size of Prochloron

The DNA base composition of the photosynthetic prokaryote Prochloron was determined (on samples collected from the natural environment) to be 40.8 mol% GC. The sharp differential melting curve indicated the absence of significant quantities of contaminating DNA from other organisms. The genome size, estimated from the renaturation kinetics of thermally denatured DNA, was 3.59×10⁹ daltons mol. wt, similar to that of many other prokaryotes. The fact that Prochloron has not yet been cultured in the laboratory cannot, therefore, be attributed to a reduced genetic information content.     

CYTOCHEMICAL STUDIES ON PROCHLOROPHYTES: LOCALIZATION OF DNA AND RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE-OXYGENASE1

We studied the distribution of the DNA-containing region and the ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCo) content of polyhedral bodies in three different prochlorophyte cell types in a search for broad evolutionary affinities of these chlorophyll b-containing prokaryotes. DNA was localized by DAPI staining and electron microscopy utilizing monoclonal anti-DNA antibody 2C-10 plus a secondary antibody labeled with colloidal gold. Antibodies against the large RuBisCo subunit from a higher plant raised in rabbits were used to localize RuBisCo in polyhedral bodies. We studied Prochloron Lewin cells from two different didemnid ascidian hosts (Lissoclinum patella and Didemnum molle) collected in Palau, West Caroline Islands, and cells of Prochlorothrix hollandica Burger-Wiersma, Stal, and Mur grown in laboratory culture. Cells of the blue-green alga Anabaena 7120 were studied for comparison. The DNA distribution was markedly different in the two Prochloron cell types. The thylakoids in cells from L. patella were concentrically arranged around a large central vacuole; the DNA-containing stromal areas appeared in thin sections as a concentric arcs between the thylakoid stacks. The central vacuole was lacking in cells from D. molle, and the thylakoid stacks and strands of DNA-containing stroma showed a more haphazard arrangement. In the filamentous Prochlorothrix the DNA-containing stroma was largely limited to a central nucleoid structure running the length of the cell. Although the DNA arrangements in Prochloron might be considered “chloroplast-like” since DNA-containing stroma is distributed, as in chloroplasts, in scattered sites among photosynthetic membranes, this is not so in Prochlorothrix, where there is an axial nucleoid, as in many filamentous cyanobacteria. Our anti-RuBisCo antibodies were selectively bound to the polyhedral bodies of all three cell types, indicating that Prochloron and Prochlorothrix, like many other autotrophic prokaryotes, possess typical carboxysomes.     

CYANOBACTERIAL EVOLUTION AND PROCHLOROPHYTE DIVERSITY AS SEEN IN DNA-DEPENDENT RNA POLYMERASE GENE SEQUENCES1

Nucleotide sequence data from DNA-dependent RNA polymerase (rpoC) genes were used to examine the phylogenetic relationships among the phycobiliprotein- and three known chlorophyll b-containing (prochlorophyte) cyanobacteria. The phylogenetic trees obtained confirm the polyphyletic nature of the prochlorophytes. Data from Prochloron cells obtained from six different tunicate host species suggest that at least two closely related groups of Prochloron exist in the same area in Palau, West Caroline Islands. Overall, however, the genetic diversity within the analyzed samples was much smaller than within the nonsymbiotic Prochlorococcus.     

Prevalence,complete genome,and metabolic potentials of a phylogenetically novel cyanobacterial symbiont in the coral-killing sponge,Terpios hoshinota

Terpios hoshinota is an aggressive, space-competing sponge that kills various stony corals. Outbreaks of this species have led to intense damage to coral reefs in many locations. Here, the first large-scale 16S rRNA gene survey across three oceans revealed that bacteria related to the taxa Prochloron, Endozoicomonas, SAR116, Ruegeria, and unclassified Proteobacteria were prevalent in T. hoshinota. A Prochloron-related bacterium was the most dominant and prevalent cyanobacterium in T. hoshinota. The complete genome of this uncultivated cyanobacterium and pigment analysis demonstrated that it has phycobiliproteins and lacks chlorophyll b, which is inconsistent with the definition of Prochloron. Furthermore, the cyanobacterium was phylogenetically distinct from Prochloron, strongly suggesting that it should be a sister taxon to Prochloron. Therefore, we proposed this symbiotic cyanobacterium as a novel species under the new genus Candidatus Paraprochloron terpiosi. Comparative genomic analyses revealed that ‘Paraprochloron’ and Prochloron exhibit distinct genomic features and DNA replication machinery. We also characterized the metabolic potentials of ‘Paraprochloron terpiosi’ in carbon and nitrogen cycling and propose a model for interactions between it and T. hoshinota. This study builds a foundation for the study of the T. hoshinota microbiome and paves the way for better understanding of ecosystems involving this coral-killing sponge.     

The relatedness of Prochloron sp. Isolated from different didemnid ascidian hosts

The relationship of Prochloron sp. isolated from four different didemnid ascidian hosts, namely Lissoclinum patella, Lissoclinum voeltzkowi, Diplosoma virensand Trididemnum cyclops was elucidated by comparative analysis of their 16S ribosomal ribonucleic acid (RNA). The oligonucleotide catalogues of the 16S rRNA obtained are almost identical, indicating a very close relationship among the prochlorophytes investigated. Phylogenetically Prochloron is a member of Cyanobacteriales.     

Contents of ultraviolet-absorbing substances in two color morphs of the photosymbiotic ascidian Didemnum molle

The contents of mycosporine-like amino acids (MAAs) were compared in the two color morphs (dark-gray and brown colonies) of the tropical ascidian Didemnum molle (Herdman, 1886), which harbors the photosymbiotic prokaryote Prochloron. The colonies of each color morph were exclusively distributed in shallow reef lagoons at the different sites. Spectroscopic and chromatographic analyses showed that the Prochloron cell density and MAA concentration in the dark-gray colonies were an estimated 1.4 and 2.4 times higher, respectively, than in the brown colonies. The significant difference in MAA contents between the color morphs was primarily due to the difference in shinorine contents (p < 0.01, Mann–Whitney U-test). The high concentration of MAAs in the dark-gray colonies may provide better conditions for Prochloron cells, compared to the brown colonies with lower MAA concentrations.     

Photoadaptation and Protection against Active Forms of Oxygen in the Symbiotic Procaryote Prochloron sp. and Its Ascidian Host

Superoxide dismutase, ascorbate peroxidase, and catalase activities were studied in the symbiotic photosynthetic procaryote Prochloron sp. and its ascidian host Lissoclinum patella. The protein-specific activities of these antioxidant enzymes in the Prochloron sp. and L. patella collected at different depths from the Great Barrier Reef, Australia, were directly proportional to irradiance, whereas the pigment concentrations in the Prochloron sp. were inversely proportional to irradiance. The presence of a cyanide-sensitive superoxide dismutase, presumably a Cu-Zn metalloprotein, in the Prochloron sp. extends the possible phylogenetic distribution of this protein. The concentration of UV-absorbing mycosporine-like amino acids is inversely proportional to irradiance in both the host and symbiont, suggesting that these compounds may not provide sufficient protection against UV radiation in high-irradiance environments. The significant differences in the specific activities of these antioxidant enzymes, cellular photosynthetic pigment concentrations, and UV-absorbing compounds from high- and low-irradiance habitats constitute an adaptive response to different photic environments. These photoadaptive responses are essential to prevent inhibition of photosynthesis by high fluxes of visible and UV radiation.     

No evidence of past bottlenecks in two Danish mustelids: results of craniometric and genetic studies in time and space

A craniometric and molecular genetic investigation was conducted in Danish stoat (Mustela erminea) and weasel (Mustela nivalis) populations. Specimens used were collected over a wide time span (stoat: 1864–2002; weasel: 1863–1990) and from several geographical regions (Jutland peninsula and the two islands of Funen and Zealand). The study was made with a temporal and a spatial perspective, allowing the estimation of differences in genetic diversity and craniometrical trait means between geographical regions and through time with the use of ancient DNA techniques. Univariate statistics of 11 trait lengths did not reveal geographical differentiation in size and shape among the different regions for the stoat, but a geographical differentiation in shape was found for the weasel. There was evidence for reductions in skull size with the year of collection in male stoats, but not in females, which suggests that some selective pressures or environmental factors have affected male stoats to a greater extent than female stoats and the weasel. Relatively high values of heterozygosity were found in both the stoat and weasel, using microsatellite markers. The level of genetic variability of the stoat collected recently was compared with the level of genetic variability in the historical samples, demonstrating that the stoat has not suffered severe loss of genetic variability through the investigated period. A comparison of recent and historical genetic variability of the weasel was not possible because the ancient DNA extracted from the weasels was too degraded. Pairwise F_ST values and assignment tests showed small but significant genetic differentiation between the different geographical regions for both the stoat and weasel. No genetic differentiation between the recent and historical samples of the stoat was found. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 88 , 541–553.     

Muramic acid in the cell walls of Prochloron

Muramic acid has been detected in Prochloron with the aid of two different techniques. It was assayed by cleaving D-lactate from muramic acid and then reducing NAD with D-lactate dehydrogenase and measuring the NADH with bacterial luciferase. Gas-liquid chromatography of trimethylsilyl derivatives of cell extracts confirmed that muramic acid was present in about the quantity given by the D-lactate assay. The amount of muramic acid present was 1.7±0.2 g/mg dry weight or 1.3fg/m² of cell surface. This suggests that the thickness of the peptidoglycan layer in Prochloron is similar to that in blue-green algae.Abbreviations D-LDH d-lactate dehydrogenase - MA muramic acid - TMS trimethylsilyl - TLE thin layer electrophoresis - GLC gas-liquid chromatography     

Application of loop-mediated isothermal amplification (LAMP)-based technology for authentication of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don

In this study, loop-mediated isothermal amplification (LAMP)-based molecular marker was developed for authentication of Catharanthus roseus, a medicinal plant. Samples of this plant were collected from different geographical locations in India. Random amplified polymorphic deoxyribonucleic acid (DNA) analysis of collected samples was carried out with 25 random primers. A 610-bp DNA fragment, common to all accessions, was eluted, cloned, and sequenced. Four LAMP primers were designed on the basis of sequence of 610 bp DNA fragment. LAMP reaction, containing 10× Bst DNA polymerase reaction buffer, Bst DNA polymerase, four in-house designed primers, dNTPs, MgSO₄, and betaine, was incubated at 65°C for 1 h. The resulting amplicon was visualized by adding SYBR Green I to the reaction tube. The data showed confirmatory results. Since the assay method is simple, sensitive, and cost-effective, it is a feasible method for identifying and authentication of C. roseus.     

Colombian Trypanosoma cruzi major genotypes circulating in patients: Minicircle homologies by cross-hybridization analysis

Here we present compelling evidence of Trypanosoma cruzi genotypes infecting 77 human cases of Chagas disease in Santander Department of Colombia. The patients were clinically studied and classified according to the presence of cardiac symptoms. We describe the distribution of the major T. cruzi genotypes circulating in this area by means of direct PCR analysis of blood samples. PCR was directed to minicircles and amplified DNAs were hybridized using genotype-specific DNA probes. These samples were previously genotyped with miniexon, 24 α rRNA and cytochrome oxidase subunit II (COII) markers. Minicircle DNA analyses were more sensitive than miniexon, 24 α rRNA and CO II genes in detecting infective T. cruzi II (Tc II). Two Tc II genotypes were identified by hybridization using two complementary DNA probes in 27.3% of the patients, with 15.3% using all three markers. These corresponded to 10 cases genotyped only by hybridization. The lineage Tc I, determined by hybridization, was the most prevalent singly or combined with different genotypes (72.7%), and at least three different T. cruzi genotypes were identified. Attempts to find two T. cruzi genotypes Tc I and Tc II in other endemic areas of Colombia revealed that one similar to the most prevalent Tc I genotype was detected in distant geographical areas. A similar Tc II genotype was found in Bolivia and Chile, revealing the great distribution of some ancestral T. cruzi genotypes. We did not detect any association between infective Tc I and Tc II lineages and the severity of the patients’ cardiac symptoms.     

Discrimination and Characterization of Two Mediterranean Species from the Laurencia Complex (Rhodomelacea) Using an NMR‐Based Metabolomic Approach

Generic and specific determination among the Laurencia complex is a challenging task. DNA barcoding combined with phenotypic investigations are mandatory for species differentiation. In this study, two morphologically different members of the Laurencia complex were investigated using untargeted ¹H‐NMR‐based metabolomics. Twenty‐one population samples were collected in order to evaluate both temporal and geographical homogeneity. Data obtained from ¹H‐NMR analysis followed by statistical analysis allowed a clear separation of all the samples into two groups. DNA mitochondrial tests confirmed this pattern and identified the two species as Laurenciella sp. and Laurencia obtusa. In addition, metabolites responsible of this discrimination were investigated directly in crude extracts by ¹³C‐NMR using an in‐house computer‐assisted method. The combination of both untargeted (¹H) and targeted (¹³C) NMR‐based metabolomic approaches proves to be a powerful and complementary approach to discriminate species from the Laurencia complex.     

Lack of relationship between gliding cyanobacteria and filamentous gliding heterotrophic eubacteria: comparison of 16S rRNA catalogues of Spirulina,Saprospira, Vitreoscilla,Leucothrix, and Herpetosiphon

Spirulina platensis and Spirulina maxima were characterized by 16S ribosomal RNA oligonucleotide cataloguing. Both species are highly related. They fall into the main group of the cyanobacteria, showing a remote relationship to Nostoc, Fischerella, Aphanocapsa, and also to Prochloron. Low similarity coefficients were found between the Spirulina species and certain organotrophic and sulfide oxidizing bacteria, viz. Saprospira grandis, Vitreoscilla stercoraria, Leucothrix mucor, Herpetosiphon aurantiacus, and Beggiatoa leptomitiformis, respectively. This result does not support the classical hypothesis that certain filamentous gliding bacteria are apochlorotic cyanobacteria.     

Partial sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase and the phylogeny of Prochloron and Prochlorococcus (Prochlorales)

The prochlorophytes, oxygenic photosynthetic prokaryotes having no phycobiliprotein but possessing chlorophylls a and b, have been proposed to have a common ancestry with green chloroplasts, yet this is still controversal. We report here that partial sequence comparisons of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, including sequence data from two prochlorophytes, Prochlorococcus and Prochloron, indicate that Prochlorococcus is more closely related to a photosynthetic bacterium, Chromatium vinosum (-purple bacteria), than to cyanobacteria, while Prochloron is closely related to the prochlorophyte Prochlorothrix and to cyanobacteria. The molecular phylogenetic tree indicates that a common ancestor of Prochlorococcus and -purple bacteria branched off from the land plant lineage earlier than Prochloron, Prochlorothrix, and cyanobacteria.Correspondence to: A. Shimada     

Prochlorococcus marinus nov. gen. nov. sp.: an oxyphototrophic marine prokaryote containing divinyl chlorophyll a and b

Several years ago, prochlorophyte picoplankton were discovered in the N. Atlantic. They have since been found to be abundant within the euphotic zone of the world's tropical and temperate oceans. The cells are extremely small, lack phycobiliproteins, and contain divinyl chlorophyll a and b as their primary photosynthetic pigments. Phylogenies constructed from DNA sequence data indicate that these cells are more closely related to a cluster of marine cyanobacteria than to their prochlorophyte relatives Prochlorothrix and Prochloron. Several strains of this organism have recently been brought into culture, and herewith are given the name Prochlorococcus marinus.     

Prochloron (Prochlorophyta): Biochemical Contributions to the Chlorophyll and RNA Composition

The prochlorophyte Prochloron, a symbiont of the colonial ascidianDidemnum molle, was collected in the Indian Ocean around Giravaru(Maldives) in depths between 1 and 40 m. The chlorophyll a tob ratio of the algal symbionts was higher in colonies livingbetween 1–6 m, compared to that determined for Prochloronfrom a depth of 30 m. This property for chromatic adaptationin correlation with changes in the total content of chlorophyllis dependent upon environmental factors. The association betweenDidemnum and Prochloron is only a facultative symbiosis. Thesize of the colonies, growing near the water surface is large(up to 3 cm), and it gradually decreases to 0.2 cm in a depthof 30 m dim locations. At a depth of 40 m the tunicates do notcontain the algal symbionts. Applying quantitative preparative isolation and sensitive immunologicalas well as biochemical detection techniques we have no evidencefor the existence of poly(A) stretches in RNA species from Prochloron.Moreover, we failed to detect both sn/scRNAs and their proteins,typically associated with them in RNP complexes from eukaryotes.From the data we suggest that mRNA synthesis proceeds in Prochloronin a way similar to prokaryotes. ¹ This contribution is dedicated to Prof. Dr. Dr. B. Schmidton the occasion of his 50th birthday.     

Photosynthesis of Prochloron as Affected by Environmental Factors

The effects of light intensity, pH, temperature, and UV irradiation on the photosynthetic rate of Prochloron isolated from the ascidian host Lissoclinum patella, collected from Palau, were examined. Photosynthesis increased with light intensity with saturation at 500 μmol/m² per second. It was maximum at pH 8 to 9 but almost completely suppressed below pH 7. The optimum temperature was 35° to 40°C, but the photosynthesis was absent at ≤20°C and at 45°C. It was recovered when the symbiont was transferred from 1 hour of incubation at ≤20°C to 35°C but not when transferred from incubation at 45°C. Ultraviolet irradiation severely inhibited the photosynthesis of Prochloron in isolation but not in vivo. This protection was brought about by the tunic covering the ascidian colony, which contains UV-absorbing mycosporine-like amino acids. These results indicate that the characteristic condition of the tropical marine environment largely determines the ecological distribution of Prochloron, and the ascidian tunic protects the organism from UV radiation. Received February 17, 2000; accepted August 8, 2000.     

Multiple ice‐age refugia in Pacific cod,Gadus macrocephalus

Pleistocene ice‐ages greatly influenced the historical abundances of Pacific cod, Gadus macrocephalus, in the North Pacific and its marginal seas. We surveyed genetic variation at 11 microsatellite loci and mitochondrial (mt) DNA in samples from twelve locations from the Sea of Japan to Washington State. Both microsatellite (mean H = 0.868) and mtDNA haplotype (mean h = 0.958) diversities were large and did not show any geographical trends. Genetic differentiation between samples was significantly correlated with geographical distance between samples for both microsatellites (F_ST = 0.028, r² = 0.33) and mtDNA (F_ST = 0.027, r² = 0.18). Both marker classes showed a strong genetic discontinuity between northwestern and northeastern Pacific populations that likely represents groups previously isolated during glaciations that are now in secondary contact. Significant differences appeared between samples from the Sea of Japan and Okhotsk Sea that may reflect ice‐age isolations in the northwest Pacific. In the northeast Pacific, a microsatellite and mtDNA partition was detected between coastal and Georgia Basin populations. The presence of two major coastal mtDNA lineages on either side of the Pacific Ocean basin implies at least two ice‐age refugia and separate postglacial population expansions facilitated by different glacial histories. Northward expansions into the Gulf of Alaska were possible 14–15 kyr ago, but deglaciation and colonization of the Georgia Basin probably occurred somewhat later. Population expansions were evident in mtDNA mismatch distributions and in Bayesian skyline plots of the three major lineages, but the start of expansions appeared to pre‐date the last glacial maximum.     

Characterization and comparison of microbial community of different typical Chinese liquor Daqus by PCR-DGGE

Aims: To identify and compare microbiota in Chinese liquor Daqu, which were produced in the different regions using different production process. Methods and Results: The DNA exacted from Daqu samples was used as a template for PCR with universal primers of 16S rRNA, 26S rRNA and 18S rRNA, respectively. The amplicons were analysed using denaturing gradient gel electrophoresis (DGGE). It was observed that the bacterial DGGE profile indicated high diversity and predominance of lactic acid bacteria. The results showed that Saccharomycopsis fibuligera and Pichia anomal were dominant yeast species and that several non‐Saccharomyces yeasts including Hanseniaspora guilliermondii, Debaryomyces hansenii, Issatchenkia orientalis and Trichosporon asahii were also detected. As for fungal DGGE, Aspergillus oryzae and Absidia blakesleeana were the most common species amongst different samples. Based on the DGGE analysis, a few differences in community structure were found between Daqu samples. Conclusions: A variety of bacteria, yeast and moulds were identified in Daqu samples, in addition to the present knowledge obtained mainly through the traditional culture‐dependent methods. Moreover, production temperature played a more decisive role on the formation of micro‐organism composition in Daqu than geographical region. Significance and Impact of the Study: PCR–DGGE technique was used in this study to fully observe and asses all microbial community (including bacteria, yeast and mould) in Chinese liquor Daqu for the first time and proved to be effective in profiling Daqu microbial diversity.