New species in the genus <Emphasis Type="Italic">Francisella</Emphasis> (Gammaproteobacteria; Francisellaceae); <Emphasis Type="Italic">Francisella piscicida</Emphasis> sp. nov. isolated from cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>)

A Francisella strain, GM2212, previously isolated from moribund farmed Atlantic cod (Gadus morhua) in Norway, is closely related to Francisella philomiragia among Francisella spp. according to its complete 16S rDNA, 16S-23S intergenic spacer, 23S rDNA, 23S–5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and hypothetical lipoprotein (LpnB) sequences. A comparison between GM2212 and the type strain of Francisella philomiragia were performed by DNA–DNA hybridization and fatty acid analysis. The DNA–DNA hybridization showed a 70% similarity. The fatty acid analysis showed only minor differences between the Francisella isolates. Due to the inconclusive result from the DNA–DNA hybridisation, major emphasis concerning the status of this isolate is made on previously published molecular, phenotypic and biochemical characters. All characteristics taken together support the establishment of GM2212 as a novel species, for which the name Francisella piscicida sp. nov. is proposed (=CNCM I-3511^T = DSM 18777^T = LMG registration number not yet available).     

Francisella sp. (Family Francisellaceae) causing mortality in Norwegian cod (Gadus morhua) farming

In 2004, a new disease was detected in cod (Gadus morhua) in western Norway. Affected cod had white granulomas in the visceral organs and skin. A species of Francisella was isolated on blood agar plates from moribund cod. The bacterium could be grown at temperatures ranging from 6 to 22°C, but did not grow at 37°C. Challenge experiments showed that Francisella sp. was the cause for the new disease. The 16S rDNA gene sequence from Francisella sp. showed 99.17% similarity to F. philomiragia, and the 16S–23S ribosomal RNA intergenic spacer (249 nt), shows a similarity with that from Francisella isolated from tilapia and F. tularensis of 96.8 and 35.9%, respectively. The 23S sequence is more similar to F. tularensis, 97.7% (2,862 nt), compared to the tilapia isolate 96.8% (2,131 nt). The partial putative outer membrane protein (FopA) sequence (781 nt) from Francisella sp. shows a similarity with that from F. tularensis and F. philomiragia of 77.3 and 98.2%, respectively. Based on sequence data, culturing temperatures and pathogenicity for cod, it is suggested that this Francisella sp. from cod could be a new species of Francisella, Family Francisellaceae.     

Systems approach to investigating host-pathogen interactions in infections with the biothreat agent <Emphasis Type="Italic">Francisella</Emphasis>. Constraints-based model of <Emphasis Type="Italic">Francisella tularensis</Emphasis>

Background  

Francisella tularensis is a prototypic example of a pathogen for which few experimental datasets exist, but for which copious high-throughout data are becoming available because of its re-emerging significance as biothreat agent. The virulence of Francisella tularensis depends on its growth capabilities within a defined environmental niche of the host cell.     

Four novel <Emphasis Type="Italic">Candida</Emphasis> species in the <Emphasis Type="Italic">Candida albicans</Emphasis>/<Emphasis Type="Italic">Lodderomyces elongisporus</Emphasis> clade isolated from the gut of flower beetles

Flower-visiting beetles belonging to three species of Cetoniidae were collected on three mountains near Beijing, China, and yeasts were isolated from the gut of the insects collected. Based on the 26S rDNA D1/D2 domain and internal transcribed spacer (ITS) region sequence analysis and phenotypic characterization, four novel anamorphic yeast species located in the Candida albicans/Lodderomyces elongisporus clade were identified from 18 of the strains isolated. The new species and type strains are designated as Candida blackwellae AS 2.3639^T (=CBS 10843^T), Candida jiufengensis AS 2.3688^T (=CBS 10846^T), Candida oxycetoniae AS 2.3656^T (=CBS 10844^T), and Candida pseudojiufengensis AS 2.3693^T (=CBS 10847^T). C. blackwellae sp. nov. was basal to the branch formed by C. albicans and C. dubliniensis with moderately strong bootstrap support. The closest relative of C. oxycetoniae was L. elongisporus. C. jiufengensis sp. nov. and C. pseudojiufengensis sp. nov. were closely related with each other and formed a branch in a subclade represented by C. parapsilosis and L. elongisporus.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>)

Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l⁻¹ phosphinothricin (PPT) and 150 mg l⁻¹ kanamycin, respectively, for shoot induction and 1 mg l⁻¹ PPT and 125 mg l⁻¹ kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T₀ and T₁ plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T₀ and T₁ transgenic plants.     

Phylogenetic analysis of the <Emphasis Type="Italic">lux</Emphasis> operon distinguishes two evolutionarily distinct clades of <Emphasis Type="Italic">Photobacterium leiognathi</Emphasis>

The luminous marine bacterium Photobacterium mandapamensis was synonymized several years ago with Photobacterium leiognathi based on a high degree of phenotypic and genetic similarity. To test the possibility that P. leiognathi as now formulated, however, actually contains two distinct bacterial groups reflecting the earlier identification of P. mandapamensis and P. leiognathi as separate species, we compared P. leiognathi strains isolated from light-organ symbiosis with leiognathid fishes (i.e., ATCC 25521^T, ATCC 25587, lequu.1.1 and lleuc.1.1) with strains from seawater originally described as P. mandapamensis and later synonymized as P. leiognathi (i.e., ATCC 27561^T and ATCC 33981) and certain strains initially identified as P. leiognathi (i.e., PL-721, PL-741, 554). Analysis of the 16S rRNA and gyrB genes did not resolve distinct clades, affirming a close relationship among these strains. However, strains ATCC 27561^T, ATCC 33981, PL-721, PL-741 and 554 were found to bear a luxF gene in the lux operon (luxABFE), whereas ATCC 25521^T, ATCC 25587, lequu.1.1 and lleuc.1.1 lack this gene (luxABE). Phylogenetic analysis of the luxAB(F)E region confirmed this distinction. Furthermore, ATCC 27561^T, ATCC 33981, PL-721, PL-741 and 554 all produced a higher level of luminescence on high-salt medium, as previously described for PL-721, whereas ATCC 25521^T, ATCC 25587, lequu.1.1 and lleuc.1.1 all produced a higher level of luminescence on low-salt medium, a characteristic of P. leiognathi from leiognathid fish light organs. These results demonstrate that P. leiognathi contains two evolutionarily and phenotypically distinct clades, P. leiognathi subsp. leiognathi (strains ATCC 25521^T, ATCC 25587, lequu.1.1 and lleuc.1.1), and P. leiognathi subsp. mandapamensis (strains ATCC 27561^T, ATCC 33981, PL-721, PL-741 and 554).Electronic Supplementary Material Supplementary material is available for this article at .     

Rapid differentiation of <Emphasis Type="Italic">Francisella</Emphasis> species and subspecies by fluorescent in situ hybridization targeting the 23S rRNA

Background  

Francisella (F.) tularensis is the causative agent of tularemia. Due to its low infectious dose, ease of dissemination and high case fatality rate, F. tularensis was the subject in diverse biological weapons programs and is among the top six agents with high potential if misused in bioterrorism. Microbiological diagnosis is cumbersome and time-consuming. Methods for the direct detection of the pathogen (immunofluorescence, PCR) have been developed but are restricted to reference laboratories.     

<Emphasis Type="Italic">Shewanella upenei</Emphasis> sp. nov., a lipolytic bacterium isolated from bensasi goatfish <Emphasis Type="Italic">Upeneus bensasi</Emphasis>

A Gram-staining-negative, motile, non-spore-forming and rod-shaped bacterial strain, 20-23R^T, was isolated from intestine of bensasi goatfish, Upeneus bensasi, and its taxonomic position was investigated by using a polyphasic study. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain 20-23R^T belonged to the genus Shewanella. Strain 20-23R^T exhibited 16S rRNA gene sequence similarity values of 99.5, 99.2, and 97.5% to Shewanella algae ATCC 51192^T, Shewanella haliotis DW01^T, and Shewanella chilikensis JC5^T, respectively. Strain 20-23R^T exhibited 93.1–96.0% 16S rRNA gene sequence similarity to the other Shewanella species. It also exhibited 98.3–98.4% gyrB sequence similarity to the type strains of S. algae and S. haliotis. Strain 20-23R^T contained simultaneously both menaquinones and ubiquinones; the predominant menaquinone was MK-7 and the predominant ubiquinones were Q-8 and Q-7. The fatty acid profiles of strain 20–23R^T, S. algae KCTC 22552^T and S. haliotis KCTC 12896^T were similar; major components were iso-C_15:0, C_16:0, C_16:1 ω7c and/or iso-C_15:0 2-OH and C_17:1 ω8c. The DNA G+C content of strain 20-23R^T was 53.9 mol%. Differential phenotypic properties and genetic distinctiveness of strain 20–23R^T, together with the phylogenetic distinctiveness, revealed that this strain is distinguishable from recognized Shewanella species. On the basis of the data presented, strain 20-23R^T represents a novel species of the genus Shewanella, for which the name Shewanella upenei sp. nov. is proposed. The type strain is 20–23R^T (=KCTC 22806^T =CCUG 58400^T).     

<Emphasis Type="Italic">Metarhizium taii var. chongqingensis</Emphasis> Nov., Anamorph of <Emphasis Type="Italic">Cordyceps</Emphasis><Emphasis Type="Italic">chongqingensis</Emphasis> sp. Nov. Isolated from a Low Altitude Area in Chongqing,China

Previously, a new Cordyceps species was isolated from a low altitude area in Chongqing, China, and named Cordyceps chongqingensis sp. nov. In this study, its anamorph was isolated and designated CQM1^T. It grew optimally on Czapek medium supplied with 0.5% silkworm flour and 0.5% soybean oil meal at 25°C, pH 5.0–5.5. The phenotypic and molecular characteristics were investigated for its identification and typing. Morphological observations under a microscope revealed that this anamorph of Cordyceps chongqingensis sp. nov. was a new species of Metarhizium. Moreover, it was identified as one of the variants of Metarhizium taii based on sequences of 26S rDNA D1/D2 and ITS regions, and thus named Metarhizium taii var. chongqingensis nov.     

<Emphasis Type="Italic">Bacillus berkeleyi</Emphasis> sp. nov., isolated from the sea urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis>

A bacterial strain, designated KMM 6244^T, was isolated from the sea urchin Strongylocentrotus intermedius and subjected to a polyphasic taxonomic investigation. The bacterium was found to be heterotrophic, aerobic, non-motile and spore-forming. Comparative phylogenetic analysis based on 16S rRNA gene sequencing placed the marine isolate in the genus Bacillus. The nearest neighbor of strain KMM 6244^T was Bacillus decolorationis LMG 19507^T with a 16S rRNA gene sequence similarity of 98.0%. Sequence similarities with the other recognized Bacillus species were less than 96.0%. The results of the DNA–DNA hybridization experiments revealed a low relatedness (37%) of the novel isolate with the type strain of B. decolorationis LMG 19507^T. Strain KMM 6244^T grew at 4–45°C and with 0–12% NaCl. It produced catalase and oxidase and hydrolyzed aesculin, casein, gelatin and DNA. The predominant fatty acids were anteiso-C_15:0, iso-C_15:0, anteiso-C_17:0, C_15:0, iso-C_16:0 and iso-C_14:0. The DNA G + C content was 39.4 mol%. A combination of phylogenetic, genotypic and phenotypic data clearly indicated that strain KMM 6244^T represents a novel species in the genus Bacillus, for which the name Bacillus berkeleyi sp. nov. is proposed. The type strain is KMM 6244^T (KCTC 12718^T = LMG 26357^T).     

<Emphasis Type="Italic">cis</Emphasis>-Cinnamoyl Glucoside as a Major Plant Growth Inhibitor Contained in <Emphasis Type="Italic">Spiraea prunifolia</Emphasis>

Crude extracts of the leaves of Spiraea prunifolia Sieb. showed high plant-growth-inhibiting activity comparable to that of S. thunbergii extracts. To isolate the causal compound in S. prunifolia, we performed bioassay-directed purification by monitoring the biological activity per unit weight of the organism containing the bioactive compound (total activity). We isolated 1-O-cis-cinnamoyl-β-D-glucopyranose (cis-CG) and identified it as the most important growth-inhibiting constituent in the crude extracts. We did not detect 6-O-(4′-hydroxy-2′-methylenebutyroyl)-1-O-cis-cinnamoyl-β-D-glucopyranose (cis-BCG) in S. prunifolia, though it is a major plant growth inhibitor in S. thunbergii together with cis-CG. We estimated the cis-CG content in S. prunifolia to be 3.84 mmol kg⁻¹ F.W. This amount is comparable to the cis-CG plus cis-BCG content in S. thunbergii (3.59 mmol kg⁻¹ F.W.). This indicates that S. prunifolia and S. thunbergii have equally high potential to inhibit plant growth, and cis-CG acts as the most important plant-growth inhibitor in S. prunifolia extracts.     

Azithromycin effectiveness against intracellular infections of <Emphasis Type="Italic">Francisella</Emphasis>

Background  

Macrolide antibiotics are commonly administered for bacterial respiratory illnesses. Azithromycin (Az) is especially noted for extremely high intracellular concentrations achieved within macrophages which is far greater than the serum concentration. Clinical strains of Type B Francisella (F.) tularensis have been reported to be resistant to Az, however our laboratory Francisella strains were found to be sensitive. We hypothesized that different strains/species of Francisella (including Type A) may have different susceptibilities to Az, a widely used and well-tolerated antibiotic.     

Development of Genus/Species-Specific PCR Analysis for Identification of <Emphasis Type="Italic">Carnobacterium</Emphasis> Strains

Heterofermentative lactic acid bacteria belonging to the genus Carnobacterium are currently divided into seven different species, C. piscicola, C. mobile, C. gallinarum, C. inhibens, C. divergens, C. funditum, and C. alterfunditum. 16S rDNA-targeted PCR assay was carried out for the identification of the genus Carnobacterium. In addition, type strains of all Carnobacterium species were analyzed by 16S–23S rDNA intergenic spacer analysis in comparison with type strains of phylogenetically related lactic acid bacteria. These methods enabled the identification and the discrimination among Carnobacterium species and the other phylogenetically related lactic acid bacteria. Likewise, analogous results were obtained by restriction analysis of amplified 16S rDNA performed with HaeIII and HinfI as restriction enzymes. Received: 25 July 2001 / Accepted: 19 October 2001     

<Emphasis Type="Italic">Paenibacillus camelliae</Emphasis> sp. nov., isolated from fermented leaves of <Emphasis Type="Italic">Camellia sinensis</Emphasis>

A novel bacterium, strain blls-2^T was isolated from Pu’er tea. The isolate was Gram-positive, endospore-forming motile rod that grew at 15∼42°C and pH 6.0∼10.2. The DNA G+C content was 48.3 mol%, the predominant isoprenoid quinone was MK-7, and the predominant cellular fatty acid was anteiso-C15:0 (54.2%) followed by C16:0 (15.5%) and iso-C16:0 (8.2%). The polar lipid pattern of blls-2^T was characterized by the presence of diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol. Phy-logenetic analysis based on 16S rRNA gene sequence showed that the strain was affiliated within the Paenibacillaceae. The strain was most closely related to Paenibacillus granivorans A30^T, with a similarity of 97.1%. Based on the phylogenetic and phenotypic characteristics of strain blls-2^T, the isolate is thought to represent a novel taxon in the genus Paenibacillus. The name Paenibacillus camelliae sp. nov. is proposed for the fermented tea isolate; the type strain is blls-2^T (= KCTC 13220^T= CECT 7361^T).     

Unusual intragenomic and interspecific variability of <Emphasis Type="Italic">Geobacillus</Emphasis> 16S-23S rRNA internal transcribed spacers

The aim of this study was to evaluate the inter-and intraspecific as well as intragenomic variability of Geobacillus 16S–23S rRNA internal transcribed spacers without tRNA genes and to compare these sequences with sequences bearing tRNA genes. In this study the structural analysis was performed in a unique way because the length and the sequence of the structural blocks were adjusted to fit the structure of 16S–23S rRNA internal transcribed spacers of five different Geobacillus species. Our study demonstrated the mosaic-like structure of 16S–23S rRNA internal transcribed spacers in Geobacillus. Some characteristics of these spacers of geobacilli were not previously reported for other bacteria: unusually short conserved sequence in the 5′ end region, some identical conserved blocks in both 5′ and 3′ regions of 16S–23S rRNA internal transcribed spacers, the same sequence blocks in both 16S–23S and 23S–5S rRNA intergenic spacers. Our study demonstrated quite uniform arrangement of the sequence blocks in Geobacillus thermodenitrificans. This species diverged early in the phylogenetic tree of the genus Geobacillus. For the phylogenetically recent species Geobacillus kaustophilus and Geobacillus lituanicus the low inter-and intraspecific, but high intragenomic variability, as a consequence of recent phylogenetic events, was established.     

A simple chromosomal marker can reliably distinguishes <Emphasis Type="Italic">Poncirus</Emphasis> from <Emphasis Type="Italic">Citrus</Emphasis> species

Several chromosome types have been recognized in Citrus and related genera by chromomycin A₃ (CMA) banding patterns and fluorescent in situ hybridization (FISH). They can be used to characterize cultivars and species or as markers in hybridization and backcrossing experiments. In the present work, characterization of six cultivars of P. trifoliata (“Barnes”, “Fawcett”, “Flying Dragon”, “Pomeroy”, “Rubidoux”, “USDA”) and one P. trifoliata × C. limonia hybrid was performed by sequential analyses of CMA banding and FISH using 5S and 45S rDNA as probes. All six cultivars showed a similar CMA⁺ banding pattern with the karyotype formula 4B + 8D + 6F. The capital letters indicate chromosomal types: B, a chromosome with one telomeric and one proximal band; D, with only one telomeric band; F, without bands. In situ hybridization labeling was also similar among cultivars. Three chromosome pairs displayed a closely linked set of 5S and 45S rDNA sites, two of them co-located with the proximal band of the B type chromosomes (B/5S-45S) and the third one co-located with the terminal band of a D pair (D/5S-45S). The B/5S-45S chromosome has never been found in any citrus accessions investigated so far. Therefore, this B chromosome can be used as a marker to recognize the intergeneric Poncirus × Citrus hybrids. The intergeneric hybrid analyzed here displayed the karyotype formula 4B + 8D + 6F, with two chromosome types B/5S-45S and two D/5S-45S. The karyotype formula and the presence of two B/5S-45S chromosomes clearly indicate that the plant investigated is a symmetric hybrid. It also demonstrates the suitability of karyotype analyses to differentiate zygotic embryos or somatic cell fusions involving trifoliate orange germplasm. During the submission of this paper, we analyzed 25 other citrus cultivars with the same methodology and we found that the chromosome marker reported here can indeed distinguish Poncirus trifoliata from grapefruits, pummelos, and one variegated access of Citrus, besides the previously reported access of limes, limons, citrons, and sweet-oranges. However, among 14 mandarin cultivars, two of them displayed a single B/5S-45S chromosome, whereas in Citrus hystrix D.C., a far related species belonging to the Papeda subgenus, this chromosome type was found in homozygosis. Since these two mandarin cultivars are probably of hybrid origin, we assume that for almost all commercial cultivars and species of the subgenus Citrus this B type chromosome is a useful genetic marker.     

Extensive analysis of nuclear cistron rDNA sequence of <Emphasis Type="Italic">Paracyclopina nana</Emphasis> (Cyclopoida: Cyclopettidae)

As a special reference of the nuclear cistron rDNA of cyclopoid copepods, we obtained the entire DNA sequence of a single rDNA repeat unit from Paracyclopina nana (Cyclopoida: Cyclopettidae). The genomic organization of P. nana rDNA (7,974 bp, 51.7% of GC) was observed to be 18S–ITS1–5.8S–ITS2–28S–IGS in order. Comparative analyses of the rDNA between P. nana and other copepods showed that the intergenic spacer (IGS) was highly informative and divergent, while other coding regions are relatively conserved. We detected eleven poly(T) tracts in the IGS, demonstrating the high AT content in this region. In addition, many sub-repeat sequence patterns (e.g., AG, AT, GC, CCG, TC) such as microsatellites were identified from the rDNA IGS of P. nana. In this article, we show the first complete sequence of rDNA from the order Cyclopoida, providing a better understanding of molecular characteristics in molecular taxonomy.     

<Emphasis Type="Italic">Flavobacterium koreense</Emphasis> sp. nov., <Emphasis Type="Italic">Flavobacterium chungnamense</Emphasis> sp. nov., and <Emphasis Type="Italic">Flavobacterium cheonanense</Emphasis> sp. nov., isolated from a freshwater reservoir

Taxonomic studies were performed on three strains isolated from Cheonho reservoir in Cheonan, Korea. The isolates were Gram-negative, aerobic, rod-shaped, non-motile, catalase-positive, and oxidase-positive. Colonies on solid media were cream-yellow, smooth, shiny, and circular. Phylogenetic analysis of the 16S rRNA gene sequences revealed that these strains belong to the genus Flavobacterium. The strains shared 98.6–99.4% sequence similarity with each other and showed less than 97% similarity with members of the genus Flavobacterium with validly published names. The DNA-DNA hybridization results confirmed the separate genomic status of strains ARSA-42^T, ARSA-103^T, and ARSA-108^T. The isolates contained menaqui-none-6 as the predominant menaquinone and iso-C_15:0, iso-C_15:0 3-OH, iso-Ci_15:1 G, and iso-C_16:0 3-OH as the major fatty acids. The genomic DNA G+C content of the isolates were 31.4–33.2 mol%. According to the phenotypic and genotypic data, these organisms are classified as representative of three novel species in the genus Flavobacterium, and the name Flavobacterium koreense sp. nov. (strain ARSA-42^T =KCTC 23182^T =JCM 17066^T =KACC 14969^T), Flavobacterium chungnamense sp. nov. (strain ARSA-103^T =KCTC 23183^T =JCM 17068^T =KACC 14971^T), and Flavobacterium cheonanense sp. nov. (strain ARSA-108^T =KCTC 23184^T =JCM 17069^T =KACC 14972) are proposed.     

<Emphasis Type="Italic">Salinarchaeum laminariae</Emphasis> gen. nov., sp. nov.: a new member of the family <Emphasis Type="Italic">Halobacteriaceae</Emphasis> isolated from salted brown alga <Emphasis Type="Italic">Laminaria</Emphasis>

Halophilic archaeal strains R26^T and R22 were isolated from the brown alga Laminaria produced at Dalian, Liaoning Province, China. Cells from the two strains were pleomorphic rods and Gram negative, and colonies were red pigmented. Strains R26^T and R22 were able to grow at 20–50°C (optimum 37°C) in 1.4–5.1 M NaCl (optimum 3.1–4.3 M) at pH 5.5–9.5 (optimum pH 8.0–8.5) and neither strain required Mg²⁺ for growth. Cells lyse in distilled water and the minimum NaCl concentration required to prevent cell lysis was 8% (w/v) for strain R26^T and 12% (w/v) for strain R22. The major polar lipids of the two strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and minor phosphatidylglycerol sulfate; glycolipids were not detected. Phylogenetic analyses based on 16S rRNA genes and rpoB′ genes revealed that strains R26^T and R22 formed a distinct clade with the closest relative, Natronoarchaeum mannanilyticum. The DNA G+C content of strains R26^T and R22 was 65.8 and 66.4 mol%, respectively. The DNA–DNA hybridization value between strains R26^T and R22 was 89%. The phenotypic, chemotaxonomic and phylogenetic properties suggest that the strains R26^T and R22 represent a novel species in a new genus within the family Halobacteriaceae, for which the name Salinarchaeum laminariae gen. nov., sp. nov. is proposed. The type strain is R26^T (type strain R26^T = CGMCC 1.10590^T = JCM 17267^T, reference strain R22 = CGMCC 1.10589).     

Efficient 5' ETS walking from conserved 18S rDNA sequences of the dinoflagellates <Emphasis Type="Italic">Alexandrium</Emphasis> and <Emphasis Type="Italic">Akashiwo sanguinea</Emphasis> (Dinophyceae)

An external transcribed spacer (ETS) walking PCR technique was developed for the isolation of unknown sequences adjacent to the 18S rDNA. This strategy relied on four "walking primers", which were designed to bind unknown sequences upstream from the 18S rDNA, and a specially programmed series of thermocycles. This method was successful in the isolation of the 5' ETS regions from harmful dinoflagellates, including Alexandrium affine, A. catenella, A. minutum, A. tamarense, and Akashiwo sanguinea. Mono-directional sequencing reactions revealed the PCR products to be 392–962 nucleotides in length, and the 5' ETS in these products were longer than 362 bp. These are the first such sequences available for A. sanguinea and the Alexandrium. In comparisons of the ETS sequences, genetic distance was considerably high within the Alexandrium. Furthermore, the sequences were significantly variable among the different strains of identical species: genetic distance was recorded at 0.0420 for A. tamarense strains and as less than 0.7841 within strains of A. sanguinea. The 5'-start nucleotide of 18S rDNA was variable between the two genera: the five species of Alexandrium contained a T base, and A. sanguinea contained an A base. These results demonstrate the effectiveness of the ETS walking PCR method. This method will be valuable in directional ETS walking from known regions to unknown regions, particularly concerning the boundary sequences of rRNA genes.