Molecular identification of filterable bacteria and archaea in the water of acidic lakes of northern Russia

Wetland ecosystems are the natural centers of freshwater formation in northern Russia lowland landscapes. The humic acidic waters formed in bogs feed the numerous lakes of the northern regions. One milliliter of the water in these lakes contains up to 10⁴ ultrasmall microbial cells that pass through “bacterial” filters with a pore size of 0.22 μm. The vast majority of these cells do not grow on nutrient media and cannot be identified by routine cultivation-based approaches. Their identification was performed by analysis of clone libraries obtained by PCR amplification of archaeal and bacterial 16S rRNA genes from the fraction of cells collected from water filtrates of acidic lakes. Most of the obtained bacterial 16S rRNA gene sequences represented the class Betaproteobacteria and exhibited the highest homology of (94–99%) with 16S rRNA genes of representatives of the genera Herbaspirillum, Herminiimonas, Curvibacter, and Burkholderia. The archaeal 16S rRNA gene clone library comprised genes of Euryarchaeota representatives. One-third of these genes exhibited 97–99% homology to the 16S rRNA genes of taxonomically described organisms of the orders Methanobacteriales and Methanosarcinales. The rest of the cloned archaeal 16S rRNA genes were only distantly related (71–74% homology) to those in all earlier characterized archaea.     

<Emphasis Type="Italic">Hoeflea siderophila</Emphasis> sp. nov., a new neutrophilic iron-oxidizing bacterium

A new representative of neutrophilic iron-oxidizing bacteria was isolated from the iron-containing sediments of the brackish low-temperature iron-rich spring of the Staraya Russa Resort (Novgorod region, Russia). The cells of strain Hf1 were thin, slightly curved rods, motile by means of a single polar flagellum. The bacterium reproduced by binary division and was capable of producing rosettes. Optimal growth was observed within the pH range of 6.2–8.5 (with an optimum at 7.5), at 9–38°C (with an optimum at 30°C), and in the salinity range of 0.1–8.5% NaCl (with an optimum at 1%). The organism was a facultative anaerobe. The strain was capable of mixotrophic and organoheterotrophic growth. Fe(II) oxidation occurred under anaerobic conditions via reduction of NO₃⁻ and N₂O, or under microaerobic conditions with oxygen as an electron acceptor. According to phylogenetic analysis based on the comparison of the 16S rRNA gene sequences, the strain was closest to the organotrophic marine bacterium Hoeflea phototrophica (98.5% similarity). The level of DNA-DNA homology with the type species of the genus Hoeflea was 19%. The DNA G + C base content was 57.5 mol %. According to its phenotypic and chemotaxonomic properties, as well as to the results of phylogenetic analysis, strain Hf1 was classified into the genus Hoeflea of the family Phyllobacteriaceae, order Rhizobiales of the phylum Alphaproteobacteria as a novel species, Hoeflea siderophila sp. nov. The type strain is Hf1^T (=DSM 21587 = VKM A7094). The GenBank accession number for the 16S rRNA gene sequences of strain Hf1^T is EU670237.     

<Emphasis Type="Italic">Thermogladius shockii</Emphasis> gen. nov., sp. nov., a hyperthermophilic crenarchaeote from Yellowstone National Park,USA

A hyperthermophilic heterotrophic archaeon (strain WB1) was isolated from a thermal pool in the Washburn hot spring group of Yellowstone National Park, USA. WB1 is a coccus, 0.6–1.2 μm in diameter, with a tetragonal S-layer, vacuoles, and occasional stalk-like protrusions. Growth is optimal at 84°C (range 64–93°C), pH 5–6 (range 3.5–8.5), and <1 g/l NaCl (range 0–4.6 g/l NaCl). Tests of metabolic properties show the isolate to be a strict anaerobe that ferments complex organic substrates. Phylogenetic analysis of the 16S rRNA gene sequence places WB1 in a clade of previously uncultured Desulfurococcaceae and shows it to have ≤96% 16S rRNA sequence identity to Desulfurococcus mobilis, Staphylothermus marinus, Staphylothermus hellenicus, and Sulfophobococcus zilligii. The 16S rRNA gene contains a large insertion similar to homing endonuclease introns reported in Thermoproteus and Pyrobaculum species. Growth is unaffected by the presence of S⁰ or SO₄ ²⁻, thereby differentiating the isolate from its closest relatives. Based on phylogenetic and physiological differences, it is proposed that isolate WB1 represents the type strain of a novel genus and species within the Desulfurococcaceae, Thermogladius shockii gen. nov., sp. nov. (RIKEN = JCM-16579, ATCC = BAA-1607, Genbank 16S rRNA gene = EU183120).     

Genetic diversity of the genus <Emphasis Type="Italic">Lactobacillus</Emphasis> bacteria from the human gastrointestinal microbiome

The species and strain genetic diversity of bacterial cultures belonging to the genus Lactobacillus, which were isolated from the gastrointestinal microbiome of the human population living in the former Soviet Union in the years 1960–1980, was studied. The bacteria demonstrated probiotic characteristics. Phylogenetic analysis of sequences of the gene coding for 16S rRNA detected earlier by us, showed that the gene found in bacteria isolated from the intestinal content of healthy adultsand represented by species L. plantarum, L. helveticus, L. casei/paracasei, L. rhamnosus, and L. fermentum has high homology (97–100%) with this gene in type representatives of the species. The genotypic and strain diversity of cultures was studied using RAPD-PCR and nonspecific primers. This method with the use of the ERIC-1 primer gave reliable and reproducible results as compared that using with M13 and MSP primers and allowed the identification of examined bacteria belonging to the genus Lactobacillus at the level of species and certification at the strain level.     

16S–23S rRNA Intergenic Spacer Region Variability in the Genus Frankia

16S–23S rRNA internally transcribed spacer (ITS) sequences from 53 Frankia strains were sequenced and sized from polymerase chain reaction amplification products and compiled with 14 selected 16S–23S ITS sequences from public database. Frankia genomes included two to three ITS copies lacking length polymorphism except for nine strains. No tRNA gene was encountered in this region. Frankia strains exhibited various lengths (369 to 452 nt) and a wide range of sequence similarity (35–100%) in the ITS region. The average pairwise distance varied from 0.368 (clusters 1 and 2) to 0.964 (clusters 3 and 4) and was 0.397, 0.138, 0.129, and 0.016, respectively, for cluster 4 (saprophytic non-infective/non-effective), clusters 1 and 3 (facultative symbiotic), and cluster 2 (obligate symbiotic). This suggests a gradual erosion of Frankia diversity concomitantly with a shift from saprophytic non-infective/non-effective to facultative and symbiotic lifestyle. Comparative sequence analyses of the 16S–23S rRNA intergenic spacer region of Frankia strains are not useful to assign them to their respective cluster or host infection group. Accurate assignment required the inclusion of the adjacent 16S and 23S rRNA gene fragments.     

Analysis of genetic variation in unisexual and bisexual lizard species of the genus Leiolepis from Southeast Asia

Using multilocus DNA fingerprinting with microsatellite probes (CAC)₅, (GACA)₄, (GGCA)₄, and (GATA)₄, intraspecific variation of the Southeast Asian lizards belonging to the genus Leiolepis (bisexual species Leiolepis reevesii and triploid parthenogenetic species Leiolepis guentherpetersi) was first examined. The L. guentherpetersi lizards were characterized by monophyletic DNA fingerprint profiles for the loci detected by the (GACA)₄, (GGCA)₄, and (CAC)₅ probes, in terms of intrapopulation similarity index constituting S = 0.96. This was different from the individual-specific profiles of the lizards from bisexual, presumably parental species, L. reevesii (S = 0.6; P < 0.001). Genetic homogeneity of triploid L. guentherpetersi lizards at the loci examined serves as one of the arguments for the parthenogenetic nature of this species. Genetic variability of triploid parthenogenetic species L. guentherpetersi appeared to be comparable with that reported earlier for the Caucasian rock lizards of the genus Darevskia, namely, D. dahli, D. armeniaca, and D. unisexualis (P > 0.05). The results of DNA fingerprinting analysis of the same L. guentherpetersi samples with the (GATA)₄ hybridization probe were unexpected. Variability of parthenogenetic species L. guentherpetersi at the (GATA)_n markers was remarkably higher than that at other DNA markers (S = 0.35; P = 3.08 × 10^?11), being comparable to the variation of the (GATA)_n DNA markers in bisexual species L. reevesii (P = 0.74). The reasons for high polymorphism of the (GATA)_n-containing loci in L. guentherpetersi still remain unclear. This polymorhism is probably associated with high instability of the loci, which can be revealed by means of family analysis of parthenogenetic offspring.     

Metschnikowia saccharicola sp. nov. and Metschnikowia lopburiensis sp. nov., two novel yeast species isolated from phylloplane in Thailand

A Gram-negative, facultatively anaerobic, motile by means of single polar flagellum, rod-shaped marine bacterium, designated strain E414, was isolated from sea water collected from a farming pond rearing marine shrimp Litopenaeus vannamei in Zhanjiang, Guangdong province, PRC. The strain was able to grow in the presence of 0.5–6% (w/v) NaCl (optimally in 3–6% (w/v) NaCl), between pH 6 and 9 (optimally at pH 7–8), between 15 and 37°C (optimally at 25–30°C). Phylogenetic analysis based on 16S rRNA gene sequences locate strain E414 in the vicinity of the coralliilyticus clade within the genus Vibrio. DNA–DNA relatedness data and multigene phylogenetic analysis based on the concatenated sequences of four genes (16S rRNA, rpoA, recA and pyrH) clearly differentiated strain E414 from its closest phylogenetic neighbours. Analysis of phenotypic features, including enzyme activities and utilization and fermentation of various carbon sources, further revealed discrimination between strain E414 and phylogenetically related Vibrio species. The major fatty acid components are C_16:1ω6c and/or C_16:1ω7c (27.4%), C_18:1ω7c and/or C_18:1ω6c (19.3%) and C_16:0 (18.2%). The DNA G+C content of strain E414 was 38.7 mol%. Based on phenotypic, chemotaxonomic, phylogenetic and DNA–DNA relatedness values, it can be concluded that E414 should be placed in the genus Vibrio as representing a novel species, for which the name Vibrio zhanjiangensis sp. nov. is proposed, with the type strain E414 (=CCTCC AB 2011110^T = NBRC 108723^T = DSM 24901).     

Confirmation that Thiobacillus halophilus and Thiobacillus hydrothermalis are distinct species within the γ-subclass of the Proteobacteria

Thiobacillus halophilus and Thiobacillus hydrothermalis share 98.7% similarity in 16S rRNA sequence, possess similar gross DNA composition (64.2 and 67.4 mol% G+C values, respectively), and have similar physiological properties. While this might have indicated that they were strains of a single species, DNA-DNA hybridization between the type strains of the two species showed only 59% hybridization, indicating the organisms to be different at the species level. Thiobacillus neapolitanus is the phylogenetically nearest neighbour of T. halophilus and T. hydrothermalis (91.6–92.1% similarity in 16S rRNA sequence) and is the only other Thiobacillus in the γ-subclass of the Proteobacteria that can be regarded as exclusively related to these two species. The 16S rRNA gene sequences of these three species are so different from those of the other thiobacilli in the γ-subclass that they justify recognition as a distinct phyletic group. Their comparative properties are summarized. Received: 23 February 1998 / Accepted: 23 April 1998     

Molecular evidence for the taxonomic status of three species of the <Emphasis Type="Italic">Sphyraena obtusata</Emphasis> group (Perciformes: Sphyraenidae) from East Asia

Genetic differences among the three species of Sphyraena with two gill rakers from East Asia (S. iburiensis, S. obtusata, and S. pinguis, defined recently as the S. obtusata group), were investigated using 799-bp sequences of the mitochondrial cytochrome b gene. Pairwise sequence differences within each of the three species were 0.0–0.4% (S. iburiensis), 0.0–0.4% (S. obtusata), and 0.0–0.6% (S. pinguis), respectively, pairwise sequence differences among the three species being 18.3–18.6% (S. iburiensis vs. S. obtusata), 14.9–15.4% (S. iburiensis vs. S. pinguis), and 17.6–18.3% (S. obtusata vs. S. pinguis), respectively. The extent of the latter were comparable to those among four other congeneric species, S. africana, S. forsteri, S. helleri, and S. japonica (16.0–24.5%). Phylogenetic analysis using the maximum-likelihood method indicated reciprocal monophyly of the three species, each clade being supported by a bootstrap value of 100%. These findings fully supported the taxonomic status of these species, recently elucidated by detailed morphological comparisons. In addition, the S. obtusata group formed a strongly supported clade against the four other congeneric species.     

EST and Mitochondrial DNA Sequences Support a Distinct Pacific Form of Salmon Louse, <Emphasis Type="Italic">Lepeophtheirus salmonis</Emphasis>

Nuclear deoxyribonucleic acid sequences from approximately 15,000 salmon louse expressed sequence tags (ESTs), the complete mitochondrial genome (16,148bp) of salmon louse, and 16S ribosomal ribonucleic acid (rRNA) and cytochrome oxidase subunit I (COI) genes from 68 salmon lice collected from Japan, Alaska, and western Canada support a Pacific lineage of Lepeophtheirus salmonis that is distinct from that occurring in the Atlantic Ocean. On average, nuclear genes are 3.2% different, the complete mitochondrial genome is 7.1% different, and 16S rRNA and COI genes are 4.2% and 6.1% different, respectively. Reduced genetic diversity within the Pacific form of L. salmonis is consistent with an introduction into the Pacific from the Atlantic Ocean. The level of divergence is consistent with the hypothesis that the Pacific form of L. salmonis coevolved with Pacific salmon (Onchorhynchus spp.) and the Atlantic form coevolved with Atlantic salmonids (Salmo spp.) independently for the last 2.5–11 million years. The level of genetic divergence coincides with the opportunity for migration of fish between the Atlantic and Pacific Ocean basins via the Arctic Ocean with the opening of the Bering Strait, approximately 5 million years ago. The genetic differences may help explain apparent differences in pathogenicity and environmental sensitivity documented for the Atlantic and Pacific forms of L. salmonis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Thermalkalibacillus uzonensis gen. nov. sp. nov, a novel aerobic alkali-tolerant thermophilic bacterium isolated from a hot spring in Uzon Caldera, Kamchatka

A novel thermophilic, alkali-tolerant, and CO-tolerant strain JW/WZ-YB58^T was isolated from green mat samples obtained from the Zarvarzin II hot spring in the Uzon Caldera, Kamchatka (Far East Russia). Cells were Gram-type and Gram stain-positive, strictly aerobic, 0.7–0.8 μm in width and 5.5–12 μm in length and produced terminal spherical spores of 1.2–1.6 μm in diameter with the mother cell swelling around 2 μm in diameter (drumstick-type morphology). Cells grew optimally at pH^25°C 8.2–8.4 and temperature 50–52°C and tolerated maximally 6% (w/v) NaCl. They were strict heterotrophs and could not use either CO or CO₂ (both with or without H₂) as sole carbon source, but tolerated up to 90% (v/v) CO in the headspace. The isolate grew on various complex substrates such as yeast extract, on carbohydrates, and organic acids, which included starch, d-galactose, d-mannose, glutamate, fumarate and acetate. Catalase reaction was negative. The membrane polar lipids were dominated by branched saturated fatty acids, which included iso-15:0 (24.5%), anteiso-15:0 (18.3%), iso-16:0 (9.9%), iso-17:0 (17.5%) and anteiso-17:0 (9.7%) as major constituents. The DNA G+C content of the strain is 45 mol%. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain JW/WZ-YB58^T is distantly (<93% similarity) related to members of Bacillaceae. On the basis of 16S rRNA gene sequence, physiological and phenotypic characteristics, the isolate JW/WZ-YB58^T (ATCC BAA-1258; DSM 17740) is proposed to be the type strain for the type species of the new taxa within the family Bacillaceae, Thermalkalibacillus uzoniensis gen. nov. sp. nov. The Genbank accession number for the 16S rRNA gene sequence is DQ221694.The Genbank accession number for the 16S rRNA gene sequence of strain JW/WZ-YB58^T is DQ221694.     

Anoxybacillus thermarum sp. nov., a novel thermophilic bacterium isolated from thermal mud in Euganean hot springs, Abano Terme, Italy

A novel aerobe thermophilic endospore-forming bacterium designated strain AF/04^T was isolated from thermal mud located in Euganean hot springs, Abano Terme, Padova, Italy. Strain AF/04^T was Gram-positive, motile, rod-shaped, occurring in pairs, or filamentous. The isolate grew between 55 and 67°C (optimum 65°C) and at pH 6.0–7.5 (optimum pH 7.2). The strain was aerobic and grew on maltose, trehalose, and sodium acetate as sole carbon sources. The G + C content of DNA was 53.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain AF/04^T falls within the genus Anoxybacillus. Levels of 16S rRNA gene sequence similarity between strain AF/04^T and the type strains of recognized Anoxybacillus species ranged from 95 to 99%. Chemotaxonomic data (major isoprenoid quinone–menaquinone-7; major fatty acid iso-C15:0 and anteiso-C17:0) supported the affiliation of strain AF/04^T to the genus Anoxybacillus. Based on phenotypic and chemotaxonomic characteristics, 16S rRNA gene sequence analysis and DNA–DNA hybridization data, it was proposed that strain AF/04^T (=DSM 17141^T = ATCC BAA 1156^T) should be placed in the genus Anoxybacillus as the type strain of a novel species, Anoxybacillus thermarum sp. nov.     

<Emphasis Type="Italic">Anoxybacillus rupiensis</Emphasis> sp. Nov., a novel thermophilic bacterium isolated from Rupi basin (Bulgaria)

Three strains of a novel thermophilic, strictly aerobic, Gram-positive, spore-forming hemo-organotrophic bacterium were isolated from three hot springs in the region of Rupi basin, Bulgaria as producers of amylolytic enzymes. Their 16S rRNA gene sequences (first 500 nucleotides) were very similar (99.8%). Strains were able to ferment a wide spectrum of carbohydrates such as sugars, polyols, and polysaccharides like xylan, glycogen and starch. Optimal growth was observed at 55–58°C, and pH at 6.0–6.5. Phylogenetic analysis of the whole 16S rRNA gene sequence clustered the strain R270^T with the representatives of the genus Anoxybacillus and with Geobacillus tepidamans. The G + C content of the genomic DNA was 41.7%. DNA–DNA hybridization analysis revealed low homology with the closest relatives (32.0 mol% homology to Geobacillus tepidamans). Fatty acid profile (major fatty acids iso-C15:0 and iso-C17:0) confirmed the affiliation of the strain to the genus Anoxybacillus. On the basis of the data presented here, we propose that strain R270^T, represents a new species of the genus Anoxybacillus for which, we recommend the name Anoxybacillus rupiensis sp. nov. (=DSM 17127^T = NBIMCC 8387^T). The 16S rRNA gene sequence data of a strain R270^T have been deposited in the EMBL databases under the accession number AJ879076.     

Phycicoccus ochangensis sp. nov., isolated from soil of a potato cultivation field

Two novel, Gram-positive, motile, coccal bacteria, strains L1b-b9^T and B5a-b5, were isolated from a potato cultivation field in Ochang, Korea. These isolates grew at 10–45°C, pH 5.0–10.0, and in the presence of 8% (w/v) NaCl. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The major menaquinone was MK-8(H₄) and the main cellular fatty acids were iso-C_14:0, iso-C_15:0, and anteiso-C_15:0. Polar lipids in strain L1b-b9^T consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, and an unknown glyco-amino lipid. The G+C content of genomic DNA was 73.6 mol%. A phylogenetic analysis based on 16S rRNA gene sequences showed that strains L1b-b9^T and B5a-b5 shared 99.36% similarity and formed a robust clade with the type species of the genus Phycicoccus. Strain L1b-b9^T is related most closely to Phycicoccus cremeus V2M29^T (97.52% 16S rRNA gene sequence similarity). On the basis of phylogenetic characteristics, the name Phycicoccus ochangensis sp. nov. is proposed for strain LIb-b9^T (=KCTC 19694^T =JCM 17595^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.     

Cryptic species of the gobioid paedomorphic genus <Emphasis Type="Italic">Schindleria</Emphasis> from Palau,Western Pacific Ocean

The extremely paedomorphic fish Schindleria (Gobioidei, Schindleriidae) is widely distributed in the Indo-Pacific Ocean, inhabiting coral reef lagoons. At least 21 cryptic species (Schindleria spp. 1–21) have been discovered around the Ryukyu and Ogasawara Islands. As a first step in extending the survey areas to the entire Indo-Pacific Ocean, the present study collected and examined Schindleria in Palau, located approximately 2,200 km from the Ryukyu Islands, for any cryptic diversity. Molecular phylogenetic analysis using partial mitochondrial 16S rRNA sequences of 71 specimens revealed four new cryptic species of Schindleria spp. 22–25 (Palau 1–4) in Palau.     

Phylogenetic analysis of Methanobrevibacter isolated from feces of humans and other animals

Comparative 16S rRNA gene sequence and genomic DNA reassociation analyses were used to assess the phylogenetic relationships of Methanobrevibacter fecal isolates. The 16S rRNA gene sequences of Methanobrevibacter smithii strain PS and the human fecal isolates B181 and ALI were essentially identical, and their genomic DNA reassociated at values greater than 94%. The analysis of 16S rRNA sequences of the horse, pig, cow, rat, and goose fecal isolates confirm that they are members of the genus Methanobrevibacter. They had a high degree of sequence similarity (97–98%) with the 16S rRNA gene of M. smithii, indicating that they share a common line of descent. The 16S rRNA genes of the horse and pig isolates had 99.3% sequence similarity. Sequence analysis of the 16S rRNA gene of the sheep fecal isolate showed that it formed a separate line of descent in the genus Methanobrevibacter. Genomic DNA reassociation studies indicate that the horse, pig, cow, and goose fecal isolates represent at least three new species. The horse and pig isolates were the only animal isolates that had > 70% genomic DNA reassociation and represent strains of a single species. The cow, goose, and sheep isolates had little or no genomic DNA reassociation with M. smithii or with each other. The relationship of the rat isolate to the other animal isolates was not determined. An evaluation of the relationship of 16S rRNA gene sequence similarity and genomic DNA reassociation of Methanobrevibacter and other methanogenic archaea indicated that genomic DNA reassociation studies are necessary to establish that two methanogenic organisms belong to the same species. Received: 17 November 1997 / Accepted: 16 January 1998     

<Emphasis Type="Italic">Marinobacter zhanjiangensis</Emphasis> sp. nov., a marine bacterium isolated from sea water of a tidal flat of the South China Sea

A novel Gram-negative, catalase- and oxidase-positive, non-sporulating, rod-shaped, aerobic bacterium, designated strain JSM 078120^T, was isolated from sea water collected from a tidal flat of Naozhou Island, South China Sea. Growth occurred with 1–15% (w/v) total salts (optimum, 2–4%), at pH 6.0–10.0 (optimum, pH 7.5) and at 4–35°C (optimum, 25–30°C). The major cellular fatty acids were C_18:1 ω9c, C_16:0, C_12:0 3-OH and C_16:1 ω7c. The predominant respiratory quinone was ubiquinone Q-9, and the genomic DNA G + C content was 60.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JSM 078120^T should be assigned to the genus Marinobacter, being related most closely to the type strains of Marinobacter segnicrescens (sequence similarity 98.2%), Marinobacter bryozoorum (97.9%) and Marinobacter gudaonensis (97.6%). The sequence similarities between the novel isolate and the type strains of other recognized Marinobacter species ranged from 96.7 (with Marinobacter salsuginis) to 93.3% (with Marinobacter litoralis). The levels of DNA–DNA relatedness between strain JSM 078120^T and the type strains of M. segnicrescens, M. bryozoorum and M. gudaonensis were 25.3, 20.6 and 18.8%, respectively. The combination of phylogenetic analysis, DNA–DNA relatedness, phenotypic characteristics and chemotaxonomic data supported the view that strain JSM 078120^T represents a novel species of the genus Marinobacter, for which the name Marinobacter zhanjiangensis sp. nov. is proposed. The type strain is JSM 078120^T (= CCTCC AB 208029^T = DSM 21077^T = KCTC 22280^T). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JSM 078120^T is FJ425903.     

Rates of Nucleotide Substitution in Angiosperm Mitochondrial DNA Sequences and Dates of Divergence Between Brassica and Other Angiosperm Lineages

We obtained 16 nucleotide sequences (∼1400 bp each) of the first intron of the mitochondrial (mt) gene for NADH subunit 4 (nad4) from 10 species of Brassicaceae. Using these new sequences and five published sequences from GenBank, we constructed a phylogenetic tree of the Brassicaceae species under study and showed that the rate of nucleotide substitution in the first intron of nad4 is very low, about 0.16–0.23 × 10⁻⁹ substitution per site per year, which is about half of the silent rate in exons of nad4. The ratios of substitution rates in this intron, ITS, and IGS are approximately 1:23:73, where ITS is the nuclear intergenic spacer between 18S and 25S rRNA genes and IGS is the intergenic spacer of 5S rRNA genes. A segment (335 bp) in the first intron of nad4 in Brassicaceae species that is absent in wheat was considered as a nonfunctional sequence and used to estimate the neutral rate (the rate of mutation) in mtDNA to be 0.5–0.7 × 10⁻⁹ substitution per site per year, which is about three times higher than the substitution rate in the rest of the first intron of nad4. We estimated that the dates of divergence are 170–235 million years (Myr) for the monocot–dicot split, 112–156 Myr for the Brassicaceae–Lettuce split, 14.5–20.4 Myr for the Brassica–Arabidopsis split, and 14.5–20.4 Myr for the Arabidopsis–Arabideae split. Received: 14 July 1998 / Accepted: 1 October 1998