Isolation and Phylogenetic Analysis of Halophilic Archaeon AJ6

Halophilic archaeon A J6 was isolated and purified from the Altun Mountain National Nature Reserve of the Xinjiang Uygur Autonomous Region.Strain AJ6 is a Gram-negative rod whose size is 0.2-0.6 by 1.6-4.2 μm,wherein a few cells are globular.The optimum salt concentration for its growth is 20% NaC1 and 0.6% Mg2+,and the optimum pH is 6.0-7.0.Morphological,physiological,and biochemical characteristics of strain AJ6 were observed.The 16S rRNA encoding gene (16S rDNA)sequence of strain A J6 was amplified by PCR,and its nucteotide sequence was determined subsequently."Clustalw"and"PHYLIP"software bags were used to analyze the 16S rDNA sequence;the homology was compared,and then the phylogenetic tree was established.The results indicate that strain AJ6 is a novel species of the genus Natrinema.The GenBank accession number of the 16S rDNA sequences of strain AJ6 is AY277584.     

The dominant bacteria shifted from the order “ Lactobacillales ” to Bacillales and Actinomycetales during a start-up period of large-scale, completely-mixed composting reactor using plastic bottle flakes as bulking agent

Bacterial community succession in the start-up of a large-scale, completely-mixed composting reactor was analyzed by 16S rRNA gene (16S rDNA) clone analysis and denaturing gradient gel electrophoresis (DGGE) combined with measurements of temperature, pH, moisture contents, and decomposing rate. DGGE analysis and physicochemical parameters showed that bacterial community succession occurred in four phases; (1) at the start of operation and pH decreasing period (day 0–3), (2) pH decreased and increased period (day 4–11), (3) middle term, moisture content decreasing and maximum temperature increased period (day 12–16) and (4) latter term, temperature decreasing period (day 17–24). Lactobacillus spp. and Bacillus coagulans were detected from the initial phase and middle term, respectively. 16S rDNA clone analysis showed that the dominant bacteria shifted from the order “Lactobacillales” to Bacillales and Actinomycetales. The order “Lactobacillales” was unique which may be caused by using the plastic bottle flakes (polyethylene terephthalate, PET) as bulking agent.     

“Bacillus thermoantarcticus” sp. nov., from Mount Melbourne,Antarctica: a novel thermophilic species

 A novel thermophilic Gram-positive bacillus, “Bacillus thermoantarcticus”, isolated from geothermal soil near the crater of Mount Melbourne, is described. The organism grows at an optimal temperature of 63^°C at pH 6.0, is oxidase-positive, catalase-negative and produces an exopolysaccharide, an exocellular xylanase, an intracellular alcohol dehydrogenase and exo- and endocellular α-glucosidase(s). The sequence of 16S rDNA is very similar to that of “Bacillus thermoglucosidasius”; however, the guanine-plus-cytosine (G+C) content is 8 mol% higher. The type strain is “Bacillus thermoantarcticus” (DSM 9572). Received : 3 February 1995/Accepted : 12 May 1995     

<Emphasis Type="Italic">Rubribacterium polymorphum</Emphasis> gen. nov., sp. nov., a novel alkaliphilic nonsulfur purple bacterium from an Eastern Siberian soda lake

An alkaliphilic nonsulfur purple bacterial (NPB) strain “Green” was isolated from sediments of the littoral zone of the soda lake (mineralization 22 g/l, pH 9.5) in the Barguzin River valley (Eastern Siberia). The cells of the new isolate are ovoid or polymorphic at latter stages. The photosynthetic membrane structures are of vesicular type. Bacteriochlorophyll a and carotenoids of both spheroidene and spirilloxanthin type are the photosynthetic pigments. Two light-harvesting systems (LH1 and LH2) are present. The new isolate is a photoheterotroph and a facultative aerobe. It grows well in the dark on organic substrates; anaerobic phototrophic growth is poor. The isolate is alkaliphilic with pH optimum of 8.5–9.5. The most abundant cell growth occurred at 5–40 g/l NaCl (optimum at 10 g/l) and 30 °C. The DNA G+C base content was 69.9 mol %. Analysis of 16S rRNA gene sequences revealed a 10% difference with the most closely related NPB (Rhodobacter species). Rubrimonas cliftoensis, a bacteriochlorophyll a-containing bacterium, is the closest relative (93.3% similarity). It is proposed that strain “Green” should be placed in the new genus and new species Rubribacterium polymorphum gen. nov., sp. nov. GenBank accession number: 16S rRNA-EU857676.     

Reidentification of the keratinase-producing facultatively alkaliphilic Bacillus sp. AH-101 as Bacillus halodurans

Alkaliphilic Bacillus sp. AH-101 was characterized in terms of physiological and biochemical characteristics, and 16S rDNA sequence homology and DNA–DNA hybridization analyses were performed. Phylogenetic analysis of strain AH-101 based on comparison of 16S rDNA sequences revealed that this strain is closely related to Bacillus halodurans. DNA–DNA hybridization of AH-101 and related Bacillus reference strains showed that the highest level of DNA–DNA relatedness (88%) was found between strain AH-101 and the B. halodurans type strain (DSM497). Our findings demonstrate that strain AH-101 is a member of the species B. halodurans. Received: June 10, 1999 / Accepted: August 6, 1999     

A <Emphasis Type="Italic">Magnetospirillum</Emphasis> strain WM-1 from a freshwater sediment with intracellular magnetosomes

Summary A helical shaped bacterium capable of producing magnetosomes, designated WM-1, was isolated from freshwater sediment through an improved isolated method that combined magnetic separation and the “race track” method. The strain WM-1 was Gram-negative, 0.2–0.4 μm in diameter and 3–4 μm in length. The strain WM-1 was identified as genus Magnetospirillum in the α-Proteobacteria according to the sequence analysis of the 16S rDNA, the morphology and physiological characteristics. The shape of the magnetosomes in WM-1was cuboidal by electron microscopy. Statistical analysis of WM-1 magnetosome crystals showed that the average number of magnetosomes in a WM-1 bacterium was 8 ± 3.4, and the average length was 54 ± 12.3 nm, and the average width was 43 ± 10.9 nm.     

Phylogeny of a novel “<Emphasis Type="Italic">Helicobacter heilmannii</Emphasis>” organism from a Japanese patient with chronic gastritis based on DNA sequence analysis of 16S rRNA and urease genes

“Helicobacter heilmannii” is an uncultivable spiral-shaped bacterium inhabiting the human gastric mucosa. It is larger and more tightly-coiled than H. pylori. We encountered a patient with chronic gastritis infected a “H. heilmannii”-like organism (HHLO), designated as SH6. Gastric mucosa derived from the patient was orally ingested by specific pathogen free mice. Colonization of the mice by SH6 was confirmed by electron microscopy of gastric tissue specimens. In an attempt to characterize SH6, 16S rRNA and urease genes were sequenced. The 16S rRNA gene sequence was most similar (99.4%; 1,437/1,445 bp) to HHLO C4E from a cheetah. However, the urease gene sequence displayed low similarity (81.7%; 1,240/1,516 bp) with HHLO C4E. Taxonomic analysis disclosed that SH6 represents a novel strain and should constitute a novel taxon in the phylogenetic trees, being discriminated from any other taxon, with the ability of infecting human gastric mucosa.     

Identification of the dominant bacterium of two-stage biooxidation of gold-arsenic concentrate

In the process of biooxidation at 39°C in a continuous mode of the gold-arsenic concentrate from the Olympiadinskoe deposit, which was pretreated by chemical leaching with ferric ions, by a microbial association from the BIO department reactors of the Polyus gold mining company, a bacterial culture designated as strain HT-4 was isolated. The bacterium was a spore-forming rod 0.5–0.6 × 1.4–2.0 μm with a flagellum. The optimal temperature for growth and Fe²⁺ oxidation was 55°C. The strain grew in the pH range from 1.21 to 2.10 with the optimum at pH 1.6. The organism was incapable of lithotrophic and organotrophic growth. It grew mixotrophically by Fe²⁺ oxidation in the presence of 0.02% yeast extract. The DNA G+C base content was 48.6 mol %. Based on comparative phylogenetic analysis of 1472-bp nucleotide sequences of 16S rRNA genes, strain HT-4 was classified as Sulfobacillus thermosulfidooxidans. Analysis by pulse-field gel electrophoresis revealed a unique profile of the NotI fragments of the chromosomal DNA. These results demonstrate the strain and species diversity of sulfobacilli in microbial associations involved in biooxidation of concentrates in different technological conditions. The strain “S. olympiadicus S-5” dominated in the process of biooxidation of original concentrate not treated with ferric iron, while S. thermosulfidooxidans HT-4 was predominant in biooxidation of the chemically leached concentrate.     

Obtaining and characterization of “Holospora curviuscula” and Holospora obtusa, bacterial symbionts of the macronuclei of Paramecium bursaria and Paramecium caudatum

The symbionts of the macronuclei of Paramecium bursaria and P. caudatum, “Holospora curviuscula” 02AZ16 and H. obtusa 88Ti, respectively, were obtained and investigated. The 16S rDNA nucleotide sequences of “Holospora curviuscula” were obtained for the first time. The differences in 16S rDNA (3.4%) suggest their classification within the genus Holospora. Molecular phylogenetic analysis of the symbionts revealed that these intranuclear symbionts of the ciliates belonged to the order Rickettsiales, forming within a compact cluster of related species.     

Rapid detection and high-resolution discrimination of the genus Streptomyces based on 16S–23S rDNA spacer region and denaturing gradient gel electrophoresis

As the leading source of antibiotics, Streptomyces species are the subject of widespread investigation. Many approaches have been tried to aid in the classification of Streptomyces isolates to the genus, species, and strain levels. Genetic methods are more rapid and convenient than classification methods based on phenotypic characteristics, but a method that is universal in detecting all Streptomyces yet selective in detecting only Streptomyces is needed. The highly conserved nature of the 16S rRNA gene (16S rDNA) combined with the need to discriminate between closely related strains results in analyses of ribosomal intergenic spacer (RIS) regions being more productive than analyses of 16S rRNA genes. PCR primers were designed to amplify the RIS region as well as a sufficient length of the 16S rRNA gene to enable phylogenetic analyses of Streptomyces. Improved selectivity and specificity for the amplification of RIS sequences from Streptomyces with environmental samples was demonstrated. The use of RIS–PCR and denaturing gradient gel electrophoresis (DGGE) was shown to be a convenient means to obtain unique genetic “fingerprints” of Streptomyces cultures allowing them to be accurately identified at species, and even strain classification levels. These RIS–PCR and DGGE approaches show potential for the rapid characterization of environmental Streptomyces populations.     

Isolation of DNA from the Uncultured “Candidatus Liberobacter” Species Associated with Citrus Huanglongbing by RAPD

“Candidatus Liberobacter,” the uncultured bacterium associated with citrus Huanglongbing (HLB) disease, is an α-Proteobacteria, and two species, “Candidatus L. africanum” and “Candidatus L. asiaticum,” have been characterized by sequence analysis of the 16S rDNA and β operon (rplKAJL-rpoBC) genes. These genes were isolated by PCR and random cloning of DNA from infected plants. However, this strategy is laborious and allowed selection of only three Liberobacter DNA fragments. In this paper, we described isolation of additional genes using Random Amplified Polymorphic DNA (RAPD). In total, 102 random 10-mer primers were used in PCR reactions on healthy and Liberobacter-infected plant DNA. Eight DNA bands amplified from infected plant DNA were cloned and analyzed. Six of them were found to be part of the Liberobacter genome by sequence and hybridization experiments. On these DNA fragments, four genes were identified: nusG, pgm, omp, and a hypothetical protein gene. These results indicate that RAPD can be used to clone DNA of uncultured organisms. Received: 14 September 1998 / Accepted: 6 October 1998     

Identification of the alga known as <Emphasis Type="Italic">Nannochloropsis</Emphasis> Z-1 isolated from a prawn farm in Hainan,China as <Emphasis Type="Italic">Chlorella</Emphasis>

An alga known as “Nannochloropsis”, isolated from a prawn farm in Hainan, China, has been critically investigated and identified as Chlorella, a member of the Chlorophyceae based on fatty acid composition, ultrastructure, and 18S rDNA. Cells of this alga were spherical, measured by 1–6 μm in diameter and were enclosed in thin walls of approximately 0.04 μm thickness. They contained several small mitochondria, two to three thylakoids and had no vacuoles. There were many pyrenoids in the algal cells and their thylakoid lamellae were sparse and not translucent. Many lipid droplets were present in the cytoplasm. The total lipid content of this alga was 3% per gram dry weight and its major fatty acids were C_16:0, C_18:0, C_18:1, C_18:2, C_18:3 and C_20:0. Eicosapentaenoic acid (C_20:5, EPA) was not detected. The length of its 18S rDNA sequence was 1,712 bp. 18S rDNA sequence analyses indicated that this alga was a species of Chlorella.     

Enrichment, isolation and characterization of pentachlorophenol degrading bacterium Acinetobacter sp. ISTPCP-3 from effluent discharge site

Three pentachlorophenol (PCP) degrading bacterial strains were isolated from sediment core of pulp and paper mill effluent discharge site. The strains were continuously enriched in mineral salts medium supplemented with PCP as sole source of carbon and energy. One of the acclimated strains with relatively high PCP degradation capability was selected and characterized in this study. Based on morphology, biochemical tests, 16S rDNA sequence analysis and phylogenetic characteristics, the strains showed greatest similarity with Acinetobacter spp. The strain was identified as Acinetobacter sp. ISTPCP-3. The physiological characteristics and optimum growth conditions of the bacterial strain were investigated. The results of optimum growth temperature revealed that it was a mesophile. The optimum growth temperature for the strain was 30°C. The preferential initial pH for the strain was ranging at 6.5–7.5, the optimum pH was 7. The bacterium was able to tolerate and degrade PCP up to a concentration of 200 mg/l. Increase in PCP concentration had a negative effect on biodegradation rate and PCP concentration above 250 mg/l was inhibitory to its growth. Acinetobacter sp. ISTPCP-3 was able to utilize PCP through an oxidative route with ortho ring-cleavage with the formation of 2,3,5,6-tetrachlorohydroquinone and 2-chloro-1,4-benzenediol, identified using gas chromatograph–mass spectrometric (GC–MS) analysis. The degradation pathway followed by isolated bacterium is different from previously characterized pathway.     

Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from <Emphasis Type="Italic">Staphylococcus</Emphasis> sp. strain AJ isolated from Korean salt-fermented Anchovy-<Emphasis Type="Italic">joet</Emphasis>

A novel fibrinolytic enzyme (AJ) was purified from Staphylococcus sp. strain AJ screened from Korean salt-fermented Anchovy-jeot. Relative molecular weight of AJ was determined as 26 kDa by using SDS-PAGE and fibrin zymography. Based on a 2D gel, AJ was found to consist of three active isoforms (pI 5.5–6.0) with the same N-terminal amino acid sequence. AJ exhibited optimum pH and temperature at 2.5–3.0 and 85°C, respectively. AJ kept 85% of the initial activity after heating at 100°C for 20 min on the zymogram gel. The Michaelis constant (K _m) and K _cat values of AJ towards α-casein were 0.38 mM and 19.73 s⁻¹, respectively. AJ cleaved the Aα-chain of fibrinogen but did not affect the Bβ- and γ-chains, indicating that it is an α-fibrinogenase. The fibrinolytic activity was inhibited by diisopropyl fluorophosphate, indicating AJ is a serine protease. Interestingly, AJ was very stable at acidic condition, SDS, and heat (100°C), whereas it was easily degraded at neutral and alkaline conditions. In particular, AJ formed an active homo-dimer in the pH range from 7.0 to 8.0. To our knowledge, a similar combination of acid and heat stability has not yet been reported for other fibrinolytic enzymes.     

Mating behavior of the rock catfish, Silurus lithophilus

The mating behavior of the rock catfish Silurus lithophilus (Siluriformes: Siluridae), a species endemic to the Lake Biwa system, was observed from May to July in 1989–1994 along the rocky shore of the lake's outlet, the Seta River. The mating behavior of S. lithophilus involved a certain behavioral sequence: “chasing,”“clinging,” and “enfolding” while “squeezing” by the male; and “circling” by the spawned pair. The mating behavior of this species was basically similar to that of S. biwaensis, but greatly different from that of S. asotus, which spawns in running water (in ditches). The mating behavior of S. lithophilus (and S. biwaensis) might have developed as an adaptation to lentic environments such as the shores of the large river or the lake. Received: October 25, 2000 / Revised: February 25, 2001 / Accepted: March 8, 2001     

<Emphasis Type="Italic">Anoxybacillus mongoliensis</Emphasis> sp. nov., a novel thermophilic proteinase producing bacterium isolated from alkaline hot spring,Central Mongolia

A Gram reaction positive, spore-forming, facultative anaerobic bacterium belonging to the Phylum Firmicutes, was isolated from alkaline hot (80°C, pH 9.8) spring Tsenher, Central Mongolia. The cells were rod shaped, feebly motile, peritrichously flagellated. Strain T4T was moderately thermophilic with optimum growth at 60°C. Maximum temperature for growth was between 70 and 75°C; minimum temperature for growth was between 35 and 30°C. Alkalitolerant, optimum pH for growth was 8.0; minimum pH for growth was between 5.0 and 5.5 and maximum was between 10.5 and 10.8. The growth was observed at NaCl concentrations of 0–5% (w/v) with the optimum at 0.2–0.5%. No growth was observed at 6% NaCl (w/v). Aerobically, the strain utilized proteinaceous substrates, organic acids and a range of carbohydrates including glucose, ribose, sucrose and xylose as well. Anaerobically, only glucose and sucrose were utilized. Strain T4^T produced thermostable alkaline subtilisin-like serine proteinase. The G + C content was 44.2 mol % (td). On the basis of 16S rRNA gene sequence similarity strain T4^T was shown to be closely related to the members of the genus Anoxybacillus (family Bacillaceae, class “Bacilli”). DNA-DNA hybridization data revealed that strain T4^T had only 38% relatedness to A. flavithermus and 28% relatedness to A. pushchinoensis. Based on its morphology, physiology, phylogenetic relationship and its low DNA-DNA relatedness values with validly published species of Anoxybacillus, it is proposed that strain T4^T represents a novel species Anoxybacillus mongoliensis sp. nov., with the type strain T4^T (=DSM 19169 =VKM 2407).     

Molecular cloning,expression, and cytokinin (6-benzylaminopurine) antagonist activity of peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis>) lectin SL-I

Isolation and purification of a α-methyl-mannoside specific lectin (SL-I) of peanut was reported earlier [Singh and Das (1994) Glycoconj J 11:282–285]. Native SL-I is a glycoprotein having ∼31 kDa subunit molecular mass and forms dimer. The gene encoding this lectin is identified from a 6-day old peanut root cDNA library by anti-SL-I antibody and N-terminal amino acid sequence homology to the native lectin. Nucleotide sequence derived amino acid sequence of the re-SL-I shows amino acid sequence homology with the N-terminal and tryptic digests’ amino acid sequence of the native SL-I (nSL-I). Presence of a putative glycosylation (QNPS) site and a hydrophobic adenine-binding (VLVSYDANS) site is also identified in SL-I. Homology modeling of the lectin suggests it to be an archetype of legume lectins. It is expressed as a ~30 kDa apoprotein in E. coli and has the carbohydrate specificity and secondary structure identical to its natural counterpart. The lectin SL-I inhibits cytokinin 6-benzylaminopurine (BA)-induced “delayed leaf senescence” and “cotyledon expansion”. Equilibrium dialysis revealed a single high-affinity binding site for adenine (7.6 × 10⁻⁶ M) and BA (1.09 × 10⁻⁵ M) in the SL-I dimer and thus suggesting that the cytokinin antagonist effect of SL-I is mediated by the direct interaction of SL-I with BA.The nucleotide sequence data reported here are available in the DDBJ/EMBL/GenBank databases under the Accession No. AJ585523     

<Emphasis Type="Italic">Halomonas sediminis</Emphasis> sp. nov., a new halophilic bacterium isolated from salt-lake sediment in China

A novel Gram-negative, slightly halophilic, catalase- and oxidase-positive, obligately aerobic bacterium, strain YIM-C248^T, was isolated from a sediment sample collected from a salt-lake in the Qaidam Basin in Qinghai, north-west China. Cells were non-sporulating short rods, occurring singly or as doublets, motile with peritrichous flagella. Growth occurred with 1–15% (w/v) NaCl [optimum 2–4% (w/v) NaCl], 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 ω7c, C_12:0 3-OH, cyclo C_19:0 ω8c, C_16:0 and C_16:1. The predominant respiratory quinone was Q-9 and the genomic DNA G + C content was 58.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain YIM-C248^T should be assigned to the genus Halomonas. The sequence similarities between the isolate and the type strains of members of the genus Halomonas were in the range of 92.5–97.5%. The combination of phylogenetic analysis, DNA–DNA hybridization data, phenotypic characteristics and chemotaxonomic differences supported the view that strain YIM-C248^T represents a new species of the genus Halomonas, for which the name Halomonas sediminis sp. nov. is proposed, with YIM-C248^T (=CCTCC AA 207031 = KCTC 22167) as the type strain. The GenBank/EMBL/DBBJ accession number for the 16S rRNA gene sequence of strain YIM-C248^T is EU135707.     

<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).     

The catalase gene differentiates between some strains of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> ssp. <Emphasis Type="Italic">anaerobius</Emphasis>

Staphylococcus aureus ssp anaerobius strain S10 was isolated from an outbreak of sheep abscess disease. Sequence of the catalase gene of this strain showed 99 % identity to the catalase gene (katB) sequence of the reference strain (S. aureus ssp. anaerobius strain MVF213) with mismatching of three base pairs. An important substitution located 1036 nucleotides upstream of the initiation codon from “C” in katB to “T” in the catalase gene of strain S10 originated a stop codon. The deduced protein (345 amino acids) is 105 amino acids shorter than that of katB. Partial sequence of the catalase gene of other 8 local isolates in addition to another reference strain (DSM 20714/ATCC 35844) revealed the same mutations in all local (African) strains, whereas the sequence of the reference (European) strain was typical to that of katB. Sequence of the catalase gene of S. aureus ssp. anaerobius strain S10 was deposited in GenBank under accession no. EU281993.