Anditalea andensis gen. nov., sp. nov., an alkaliphilic, halotolerant bacterium isolated from extreme alkali–saline soil

A novel alkalophilic salt-tolerant rod-shaped bacterium, designated ANESC-S^T, was isolated from an extremely alkali–saline soil in the rural area of Anda city in northeast China. Taxonomic study using a polyphasic approach revealed that this non-motile, orange colony-forming microbe was Gram-negative and obligately aerobic. Optimal growth of strain ANESC-S^T was achieved in the presence of NaCl with a concentration range of 0.5 to 4 % and pH between 7.5 and 9.2, and at temperatures ranging from 10 to 37 °C. Phylogenetic analysis of 16S rRNA sequences showed that of strain ANESC-S^T is most homologous to Mongoliicoccus roseus MIM28^T and Litoribacter ruber YIM CH208^T with sequence similarity of 95.1 and 93.2 %, respectively. The genomic DNA G+C content of strain ANESC-S^T was determined to be 39.1 mol%. The main isoprenoid quinone in ANESC-S^T was found to be menaquinone-7. The main fatty acids were found to be iso-C_15:0 (27.5 %), iso-C_17:03-OH (14.0 %), anteiso-C_15:0 (9.8 %), summed feature 9 (iso-C_17:1ω9c and/or 10-methyl C_16:0 10.6 %) and summed feature 3 (C_16:1w7c/C_16:1w6c, 9.78 %). Based on the phenotypic, chemotaxonomic and phylogenetic data, strain ANESC-S^T is considered to represent a new genus and species classified into the order Cytophagales, for which the name Anditalea andensis gen. nov., sp. nov. is proposed. The type strain is ANESC-S^T (=CICC 10485^T = NCCB 100412^T).     

Purification and characterization of a liquefying α-amylase from alkalophilic thermophilic Bacillus sp. AAH-31

α-Amylase (EC 3.2.1.1) hydrolyzes an internal α-1,4-glucosidic linkage of starch and related glucans. Alkalophilic liquefying enzymes from Bacillus species are utilized as additives in dishwashing and laundry detergents. In this study, we found that Bacillus sp. AAH-31, isolated from soil, produced an alkalophilic liquefying α-amylase with high thermostability. Extracellular α-amylase from Bacillus sp. AAH-31 (AmyL) was purified in seven steps. The purified enzyme showed a single band of 91 kDa on SDS-PAGE. Its specific activity of hydrolysis of 0.5% soluble starch was 16.7 U/mg. Its optimum pH and temperature were 8.5 and 70 °C respectively. It was stable in a pH range of 6.4-10.3 and below 60 °C. The calcium ion did not affect its thermostability, unlike typical α-amylases. It showed 84.9% of residual activity after incubation in the presence of 0.1% w/v of EDTA at 60 °C for 1 h. Other chelating reagents (nitrilotriacetic acid and tripolyphosphate) did not affect the activity at all. AmyL was fully stable in 1% w/v of Tween 20, Tween 80, and Triton X-100, and 0.1% w/v of SDS and commercial detergents. It showed higher activity towards amylose than towards amylopectin or glycogen. Its hydrolytic activity towards γ-cyclodextin was as high as towards short-chain amylose. Maltotriose was its minimum substrate, and maltose and maltotriose accumulated in the hydrolysis of maltooligosaccharides longer than maltotriose and soluble starch.     

Isolation and characterization of long-chain alkane–degrading Acinetobacter sp. BT1A from oil-contaminated soil in Diyarbakır,in the Southeast of Turkey

A strain of long-chain alkane–degrading bacteria, BT1A, was isolated from oil-contaminated soil in Diyarbak?r, in the southeast of Turkey. Morphological, biochemical, and physiological characterization and 16S rRNA gene sequence analysis showed that the strain BT1A was a member of Acinetobacter genus, and it was found to be closely related to Acinetobacter baumannii. The strain BT1A was able to utilize crude petroleum as carbon and energy sources in order to grow. Among the aliphatic hydrocarbons, growth was observed only in the medium containing long-chain alkanes (tridecane, pentadecane, and hexadecane) and squalene. Hexadecane was the most preferred hydrocarbon among the long-chain alkanes. Gas chromatography–mass spectrometry (GC-MS) analysis showed that BT1A degraded 83% of n-alkanes of 1% crude oil in 7 days. The present study indicates that the isolated strain can well be used for biodegradation of hydrocarbons in oil-contaminated sites.     

Isolation and characterization of a thermophilic bacillus strain,that degrades phenol and cresols as sole carbon source at 70 °C

A phenol-degrading thermophilic bacterium, designated Bacillus sp. A2, was isolated from a water and mud sample from a hot spring in Iceland. The aerobic isolate grew optimally on phenol at 65 °C. At 70 °C, 85% of the optimal growth rate was still observed. No growth was observed at 40 °C and 75 °C. Bacillus sp. A2 is a gram-positive spore-forming rod. According to 16S rDNA analysis Bacillus sp. A2 is closely related to Bacillus stearothermophilus, Bacillus kaustophilus and Bacillus thermoleovorans. Bacillus sp. A2 degraded phenol completely in concentrations up to 5 mM. In addition, all three isomers of cresol were utilized as sole carbon and energy sources. The degradation of phenols proceeds via the meta-cleavage pathway and the enzymes involved in its degradation are constitutively expressed. Received: 13 May 1996 / Received revision: 29 July 1996 / Accepted: 12 August 1996     

Production, purification and characterization of a constitutive intracellular α-galactosidase from the thermophilic fungus Humicola sp

The thermophilic fungus Humicola sp constitutively produces intracellular α-galactosidase (1.33 U mg⁻¹ protein) within 48 h at 45°C in shaken flasks, when grown in a medium containing 7% wheat bran extract as a carbon source and 0.5% yeast extract as a nitrogen source. The enzyme has been purified to homogeneity by ultrafiltration, ethanol precipitation, DEAE cellulose and Sephacryl S-300 chromatography with a 124-fold increase in specific activity and 29.5% recovery. The molecular weight of the enzyme is 371.5 kDa by gel filtration on Sephacryl S-300 and 87.1 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme has an optimum temperature of 65°C and an optimum pH of 5.0. Humicola α-galactosidase is a glycoprotein with 8.3% carbohydrate content and is acidic in nature with a pI of 4.0. The K _m S for p-nitrophenyl-α-D-galactopyranoside, O-nitrophenyl-α-D-galactopyranoside, raffinose and stachyose are 0.279, 0.40, 1.45 and 1.42 mM respectively. The enzyme activity was strongly inhibited by Ag⁺ and Hg²⁺. D-Galactose inhibited α-galactosidase competitively and the inhibition constant (K _i) for galactose was 11 mM. Received 28 January 1999/ Accepted in revised form 07 April 1999     

Isolation and Characterization of a Δ5-Desaturase from Oblongichytrium sp.

We isolated a cDNA clone with homology to known desaturase genes from Oblongichytrium sp., recently classified as a new genus of thraustochytrids (Labyrinthulomycetes), and found that it encoded Δ5-desaturase by its heterologous expression in yeast. The enzyme had higher activity toward 20:4n-3 than 20:3n-6, indicating that this Δ5-desaturase can be used in the production of n-3 polyunsaturated fatty acids in transgenic organisms.     

Marinifaba aquimaris gen. nov., sp. nov., a novel chitin‐degrading gammaproteobacterium in the family Alteromonadaceae isolated from seawater of the Mariana Trench

Antonie van Leeuwenhoek - A novel Gram-negative, rod-shaped, aerobic, oxidase-positive and catalase-negative bacterium, designated strain SM1970T, was isolated from a seawater sample collected from...     

Thermoactinospora rubra gen. nov., sp. nov., a thermophilic actinomycete isolated from Tengchong, Yunnan province, south–west China

Two novel Gram-positive, spore-forming, thermophilic actinomycetes, designated as strain YIM 77501^T and YIM 77570, were isolated from a sandy soil sample collected at Tengchong National Volcanic Geological Park, Yunnan province, south–west China. Phylogenetic analysis based on the 16S rRNA gene sequences suggested that the two isolates fell within the family Streptosporangiaceae. The strains formed extensively branched substrate and aerial mycelia which carried masses of long, straight or irregular spore chains composed of warty ornamented spores. Cell walls of the two strains contained meso-diaminopimelic acid and glucose, galactose, mannose and ribose were detected as whole-cell sugars. The predominant menaquinones were MK-9(H₄) and MK-9(H₆). The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, N-acetylglucosamine-containing phospholipids and phosphatidylinositol, phosphatidylinositolmannosides. The major cellular fatty acids were iso-C_16:0 and 10-methyl C_17:0. The DNA G+C content was 74–76 mol%. On the basis of the morphological and chemotaxonomic characteristics as well as the phylogenetic analysis, these strains represents a novel species of a new genus within the family Streptosporangiaceae, for which the name Thermoactinospora rubra gen. nov., sp. nov. is proposed. The type strain of T. rubra is YIM 77501^T (=DSM 45614^T = CCTCC AA 2011014^T).     

Isolation and Characterization of a Novel Endo-β-galactofuranosidase from Bacillus sp.

A soil bacterium capable of growing on a polysaccharide containing β(1→6)galactofuranoside residues derived from the acidic polysaccharide of Fusarium sp. as a carbon source has been isolated. From various bacteriological characteristics, the organism was identified as a Bacillus sp. The bacterium produced β- galactofuranosidase inductively in the culture media. The most effective inducer for the β-galactofuranosidase production was a polysaccharide containing β(1→5) or β(1→6)-linked galactofuranoside residues, but gum arabic, gum guar, gum ghati, arabinogalactam, araban, and pectic acid did not induce the enzyme. The enzyme had three different molecular weight forms. The low molecular-weight form was purified by a combination of Toyopearl HW-55 and DEAE-Toyopearl 650S column chromatographies, and preparative polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 67,000 by SDS–polyacrylamide gel electrophoresis. The enzyme was most active at pH 6 and 37°C, and was stable between pH 4 to 8 at 5°C. The action of the enzyme was inhibited by the addition of Cd²⁺, Co²⁺, Hg²⁺, Zn²⁺, iodoacetic acid, and EDT A. The purified enzyme cleaved β(1→5) and β(1→6)-linked galactofuranosyl chains. Based upon the mode of liberation of galactofuranosyl residues from pyridylamino β(1→6)-linked galactofuranoside oligomers, the enzyme can be classified as an endo-β-galactofuranosidase that randomly hydrolyzes the linkage.     

Cloning,expression and biochemical characterization of a β-carbonic anhydrase from the soil bacterium Enterobacter sp. B13

A recombinant carbonic anhydrase (CA, EC 4.2.1.1) from the soil-dwelling bacterium Enterobacter sp. B13 was cloned and purified by Co²⁺ affinity chromatography. Bioinformatic analysis showed that the new enzyme (denominated here B13-CA) belongs to the β-class CAs and to possess 95% homology with the ortholog enzyme from Escherichia coli encoded by the can gene, whereas its sequence homology with the other such enzyme from E. coli (encoded by the cynT gene) was of 33%. B13-CA was characterized kinetically as a catalyst for carbon dioxide hydration to bicarbonate and protons. The enzyme shows a significant catalytic activity, with the following kinetic parameters at 20?°C and pH of 8.3: k_cat of 4.8?×?10⁵?s^?1 and k_cat/K_m of 5.6?×?10⁷ M^?1?×?s^?1. This activity was potently inhibited by acetazolamide which showed a K_I of 78.9?nM. Although only this compound was investigated for the moment as B13-CA inhibitor, further studies may reveal new classes of inhibitors/activators of this enzyme which may show biomedical or environmental applications, considering the posssible role of this enzyme in CaCO₃ biomineralization processes.     

Description of Ulvella elegans sp. nov. and U. islandica sp. nov. (Ulvellaceae,Ulvophyceae) from Iceland – a study based on morphology of species in culture and tufA gene sequences

Two new Ulvella species, U. elegans R. Nielsen & K. Gunnarsson and U. islandica R. Nielsen & K. Gunnarsson are described. These microfilamentous marine green algae were found in the sublittoral zone in northern Iceland, epiphytic on Euthora cristata and associated with a calcareous polychaete tube, respectively. Unialgal cultures were established from field-collected material for morphological observations. In culture, Ulvella elegans was characterized by rosettes of monostromatic pseudoparenchyma consisting of radiating filaments with a margin of mutually free filaments. Each cell had one pyrenoid. Hairs were not observed. Ulvella islandica had a heterotrichous morphology, consisting of dense tufts of upright broad branches and much narrower, rhizoid-like branches. Acrochaete-type hairs occurred; these are hyaline non-septate merocytic extensions from a more or less bulbous base, which may be separated from the vegetative cell below. Most cells had one pyrenoid except for a few broad cells which had two or three. In a phylogenetic reconstruction based on the chloroplast-encoded tufA gene, the sequences for the two species were clearly distinct from any other Ulvella sequence available for this gene. Ulvella islandica was placed in a clade together with U. lens, U. wittrockii, U. reticulata and U. pseudorepens. Ulvella elegans occupied a branch deep in the phylogeny but the position was poorly supported.     

Glutamic acid 219 is critical for the thermostability of a truncated α-amylase from alkaliphilic and thermophilic Bacillus sp. strain TS-23

The functional and structural significance of glutamic acid 219 of a N- and C-terminally truncated Bacillus sp. strain TS-23 α-amylase (BACΔNC) was explored by the approach of site-directed saturation mutagenesis. The expressed wild-type and mutant enzymes have been purified by nickel-chelate chromatography and their molecular mass was determined to be approximately 54 kDa by SDS/PAGE. Except E219F, E219P, and E219W, all other mutant enzymes exhibited a lower shift in their optimum temperatures with respect to the wild-type enzyme. A decreased thermostability was also found in all of the mutant enzymes when compared with the wild-type form of BACΔNC. Except E219F, E219P, and E219W mutant enzymes, greater than 2-fold decrease in k _cat and a similar substrate affinity relative to the wild-type BACΔNC were observed for the rest mutant enzymes. Based on these observations, it is suggested that Glu-219 apparently plays an important role in the thermostability of BACΔNC.     

“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     

Purification and characterization of a novel thermostable α-l-arabinofuranosidase (α-l-AFase) from Chaetomium sp.

The purification and characterization of an extracellular α-l-arabinofuranosidase (α-l-AFase) from Chaetomium sp. was investigated in this report. The α-l-AFase was purified to homogeneity with a purification fold of 1030. The purified α-l-AFase had a specific activity of 20.6 U mg^?1. The molecular mass of the enzyme was estimated to be 52.9 kDa and 51.6 kDa by SDS–PAGE and gel filtration, respectively. The optimal pH and temperature of the enzyme were pH 5.0 and 70 °C, respectively. The enzyme was stable over a broad pH range of 4.0–10.0 and also exhibited excellent thermostability, i.e., the residual activities reached 75% after treatment at 60 °C for 1 h. The enzyme showed strict substrate specificity for the α-l-arabinofuranosyl linkage. The K_m and V_max values for p-nitrophenyl (pNP)-α-l-arabinofuranoside were calculated to be 1.43 mM and 68.3 μmol min^?1 mg^?1 protein, respectively. Furthermore, the gene encoding α-l-AFase was cloned and sequenced and found to contain a catalytic domain belonging to the glycoside hydrolase (GH) family 43 α-l-AFase. The deduced amino acid sequence of the gene showed the highest identity (67%) to the putative α-l-AFase from Neurospora crassa. This is the first report on the purification, characterization and gene sequence of an α-l-AFase from Chaetomium sp.     

Isolation and characterization of fourteen microsatellites from a tree peony (Paeonia suffruticosa)

Tree peonies are well known garden flowers and very important medicinal plants with some 1,000 cultivars. Most of the cultivars are believed to be of hybrid origin and their wild parents are suffering from severe depopulation. To rejuvenate the traditional cultivars and assess the genetic diversity of the critically endangered wild species for conservation purpose, it is crucial to use suitable molecular markers. In this study, we isolated 14 polymorphic microsatellite loci from an enriched genomic library. These loci were verified by re-cloning and re-sequencing and their characteristics were tested with 20 individuals. On average there are 5.5 alleles per locus, and mean values of observed heterozygosity and expected heterozygosity are 0.41 and 0.67, respectively.     

Purification and characterization of a thermostable λ-carrageenase from a hot spring bacterium,Bacillus sp.

Purpose of work The purpose of this study is to report a thermostable λ-carrageenase that can degrade λ-carrageenan yielding neo-λ-carrabiose at 75 °C. A thermophilic strain Lc50-1 producing λ-carrageenase was isolated from a hot spring in Indonesia and identified as a Bacillus sp. The λ-carrageenase, Cga-L50, with an apparent molecular weight of 37 kDa and a specific activity of 105 U/mg was purified from the culture supernatant. The optimum pH and temperature of Cga-L50 were 8.0 and 75 °C, respectively. The enzyme was stable from pH 6–9 and retained ~50 % activity after holding at 85 °C for 10 min. Significant activation of Cga-L50 was observed with K⁺, Ca²⁺, Co²⁺, and Na⁺; whereas, the enzyme activity was inhibited by Sr²⁺, Mn²⁺, Fe²⁺, Cu²⁺,Cd²⁺, Mg²⁺, and EDTA. Cga-L50 is an endo-type λ-carrageenase that hydrolyzes β-1,4-linkages of λ-carrageenan, yielding neo-λ-carrabiose as the main product. This study is the first to present evidence of thermostable λ-carrageenase from hot spring bacteria.     

Blastocatella fastidiosa gen. nov., sp. nov., isolated from semiarid savanna soil – The first described species of Acidobacteria subdivision 4

Acidobacteria represent abundant members of soil microbial communities but only few representatives could be isolated and validly described so far. Currently, eighteen species of subdivision 1, one species of subdivision 3, three species of subdivision 8, and one species of subdivision 10 are recognized. In contrast, Acidobacteria of subdivision 4 have largely escaped cultivation although they belong to the most abundant and diverse acidobacterial groups in soils. A member of subdivision 4, strain A2-16^T, was isolated from a semiarid savanna soil. Cells were motile spheres to rods with a tendency to form chains and larger aggregates. Cultures were orange to pink colored, neutrophilic mesophiles, and showed aerobic chemoorganoheterotrophic growth on very few complex substrates and protocatechuate, and weak growth on chitin, cellulose and starch. While protein substrates such as casamino acids or peptone were utilized, individual amino acids did not promote growth. Also, growth on alternative electron acceptors or fermentative growth could not be observed. Major fatty acids were summed features 1 (15:1 iso H/13:0 3-OH) and 3 (16:1ω7c/15:0 iso 2-OH). The major quinone was MK-8. The DNA G+C content was 46.5 mol%. Phylogenetic analysis placed A2-16^T amidst uncultured members of Acidobacteria subdivision 4. The most closely related environmental 16S rRNA gene sequences (96–97% nucleotide identity) were several clone sequences from terrestrial environments. Based on these characteristics, the isolated strain is proposed as a new species of a novel genus, Blastocatella fastidiosa gen. nov., sp. nov. The type strain of B. fastidiosa is A2-16^T (=DSM 25172^T = LMG26944^T).     

Corynebacterium antarcticum sp. nov., Corynebacterium marambiense sp. nov., Corynebacterium meridianum sp. nov., and Corynebacterium pygosceleis sp. nov., isolated from Adélie penguins (Pygoscelis adeliae)

A taxonomic study was conducted on 16 bacterial strains isolated from wild Adélie penguins (Pygoscelis adeliae) from Seymour (Marambio) Island and James Ross Island. An initial screening by repetitive sequence-based PCR fingerprinting divided the strains studied into four coherent groups. Phylogenetic analysis based on 16S rRNA gene sequences assigned all groups to the genus Corynebacterium and showed that Corynebacterium glyciniphilum and Corynebacterium terpenotabidum were the closest species with 16S rRNA gene sequence similarities between 95.4 % and 96.5 %. Further examination of the strains studied with ribotyping, MALDI-TOF mass spectrometry, comprehensive biotyping and calculation of average nucleotide identity and digital DNA–DNA hybridisation values confirmed the separation of the four groups from each other and from the other Corynebacterium species. Chemotaxonomically, the four strains P5828^T, P5850^T, P6136^T, P7210^T representing the studied groups were characterised by C_16:0 and C_18:1 ω9c as the major fatty acids, by the presence of meso-diaminopimelic acid in the peptidoglycan, the presence of corynemycolic acids and a quinone system with the predominant menaquinone MK-9(H₂). The results of this study show that the strains studied represent four new species of the genus Corynebacterium, for which the names Corynebacterium antarcticum sp. nov. (type strain P5850^T = CCM 8835^T = LMG 30620^T), Corynebacterium marambiense sp. nov. (type strain P5828^T = CCM 8864^T = LMG 31626^T), Corynebacterium meridianum sp. nov. (type strain P6136^T = CCM 8863^T = LMG 31628^T) and Corynebacterium pygosceleis sp. nov. (type strain P7210^T = CCM 8836^T = LMG 30621^T) are proposed.     

Rugiloricus bacatus sp. nov. (Loricifera ‐Pliciloricidae) and a ghost‐larva with paedogenetic reproduction

Abstract

A new species of Loricifera, Rugiloricus bacatus sp. nov. is described together with the diagnoses of two other Rugiloricus species, Rugiloricus sp. nov. A and B, from the Faroe Bank (North Atlantic). Characteristic for all three species is the presence of a new type of reduced larva, a ghost‐larva. This type of reduced larva was discovered in 1986 by Jeanne Renaud‐Mornant, but it was with the Faroe Bank material that it was first discovered that the ghost‐larvae belonged to the phylum Loricifera. The ghost‐larvae are eitherfound inside late instar Higgins‐larvae, called penultimate Higgins‐larvae, or in the sediment. The three types of Higgins‐larvae from the Faroe Bank can be distinguished by characters such as anterior setae, posterior setae and toes. The adults of Rugiloricus bacatus sp. nov. are characterised by a prominent ruff resembling a pearl necklace; two of the eight clavoscalids are modified in the 1st row; the 2nd row of leg‐shaped scalids are very large and robust, and the 9th row with 30 beak‐like scalids alternating with 30 alternating plates. The postlarvae are free‐living and their scalids on the introvert are reduced to protoscalids. Postlarvae and adult stages have not been found for Rugiloricus sp. nov. A and B and therefore only diagnoses of these two species are presented here.