Phylogenetic analysis and characterization of lipolytic activity of halophilic archaeal isolates

Five isolates designated as B45, D83A, A206A, A85 and E49 and found to possess a activity were taxonomically classified on the basis of their phylogenetic, phenotypic and chemotaxonomic characteristics. The isolates were determined to be Gram-negative, catalase and oxidase positive, hydrolyzing Tween 80 and 60 but not starch, need 3.5-4 M NaCl for optimal growth and lack of anaerobic growth with arginine or DMSO. All isolates had the highest lipolytic activity at pH 8.5. Lipase and esterase activities increased with salt concentration up to 3-4.5 M NaCl, and decreased at 5 M NaCl. Esterase and lipase showed their maximal activities at 50-55 degrees C and 60-65 degrees C, respectively. The phylogenetic tree constructed by the neighbor-joining method indicated that the strain B45 and A85 were closely related to the members of genera Halovivax and Natrinema, respectively. The closest relative of the strain A206A and D83A were found to be Haloterrigena saccharevitans. The strain E49 displayed a more distant relationship to known strains.     

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).     

Silicibacter lacuscaerulensis gen. nov., sp. nov., a mesophilic moderately halophilic bacterium characteristic of the Blue Lagoon geothermal lake in Iceland

Mesophilic, moderately halophilic bacteria were isolated from a silica-rich geothermal lake, the Blue Lagoon in Iceland. The isolates are strictly aerobic, but reduce nitrate to nitrite, and are oxidase- and catalase-positive. The nonsporeforming and nonmotile Gram negative rods are 0.6–0.8 μm in diameter and variable in length (9–18 μm), and contain gas vacuoles. The GC content in their DNA is 66.15%. The minimum, optimum, and maximum temperatures for growth are 22°C, 45°:C, and 50°C, respectively. The isolates do not grow without added salt in the medium and can grow at up to 7% NaCl (w/v). The optimal salinity for growth is 3.5%–4% NaCl. The pH range for growth is 6.5–8.5, with the optimal pH at 7.0. At optimal conditions the bacterium has a doubling time of 80 min. The main cytochrome is a membrane-bound cytochrome c with an α-peak at 549 nm. Sequencing of 16S rRNA from the type strain ITI-1157 revealed it to be a proteobacterium of the α-subclass with the closest relatives being Roseobacter litoralis and Paracoccus kocurii. The new isolates do not contain bacteriochlorophyll a and are considered to represent a new genus and a new species, Silicibacter lacuscaerulensis. Received: September 17, 1996 / Accepted: December 10, 1996     

Salt-activated endoglucanase of a strain of alkaliphilic Bacillus agaradhaerens

An endoglucanase was purified to homogeneity from an alkaline culture broth of a strain isolated from␣seawater and identified here as Bacillus agaradhaerens JAM-KU023. The molecular mass was around 38-kDa and the N-terminal 19 amino acids of the purified enzyme exhibited 100% sequence identity to Cel5A of B. agaradhaerens DSM8721^T. The enzyme activity increased around 4-fold by the addition of 0.2–2.0 M NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). KCl, Na₂SO₄, NaBr, NaNO₃, CH₃COONa, LiCl, NH₄NO₃, and NH₄Cl also activated the enzyme up to 2- to 4-fold. The optimal pH and temperature values were pH 7–9.4 and 60 °C with 0.2 M NaCl, but pH 6.5–7 and 50 °C without NaCl; enzyme activity increased approximately 6-fold at 60 °C with 0.2 M NaCl compared to that at 50 °C without NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). The thermostability and pH stability of the enzyme were not affected by NaCl. The enzyme was very stable to several chemical compounds, surfactants and metal ions (except for Fe²⁺ and Hg²⁺ ions), regardless whether NaCl was present or not. * The nucleotide sequence of 16S rRNA of this strain has been submitted to DDBJ, EMBL, and GenBank databases under accession no. AB211544.     

Growth Enhancement of Chickpea in Saline Soils Using Plant Growth-Promoting Rhizobacteria

Bacterial isolates with the ability to tolerate salinity and plant growth-promoting features were isolated from the saline areas of Gujarat, India, that is, Bhavnagar and Khambat. A total of 176 strains of rhizobacteria were isolated out of which 62 bacterial strains were able to tolerate 1 M NaCl. These were then further studied for their potential plant growth-promoting rhizobacteria characteristics like phosphate solubilization, siderophore production, and IAA production. Twenty-eight isolates of the 62 strains showed good tricalcium phosphate solubilization in solid medium in the range of 9–22 mm and 15 isolates showed good phosphate solubilization in liquid medium in the range of 9–45 μg/ml. Siderophore production was checked in all 15 isolates, and 13 were screened out that produced the hydroxamate type of siderophore in the range of 11–50 mM. Among the 13 isolates, 10 were able to produce indole acetic acid in the range of 10–26 μg/ml after 72 h of incubation. Pot trials were carried out on chickpea under 300 mM NaCl stress using the best five isolates. Plants inoculated with MSC1 or MSC4 isolates showed an increase in the parameters that evaluate plant growth when compared to uninoculated controls. Strains MSC1 and MSC4 were identified as Pseudomonas putida and Pseudomonas pseudoalcaligens, respectively, according to sequence analysis of the 16S rRNA gene.     

Halorussus rarus gen. nov., sp. nov., a new member of the family Halobacteriaceae isolated from a marine solar saltern

Two halophilic archaeal strains TBN4^T and TBN5 were isolated from Taibei marine solar saltern in Jiangsu, China. Both strains showed light red-pigmented colonies and their cells were rod, motile and Gram-stain-negative. They were able to grow at 25–50°C (optimum 37°C), at 1.4–4.3 M NaCl (optimum 2.1 M NaCl), at 0–1.0 M MgCl₂ (optimum 0.005 M MgCl₂) and at pH 6.0–9.0 (optimum pH 7.0). Their cells lyse in distilled water and minimal NaCl concentration to prevent cell lysis is 8% (w/v). The major polar lipids of the two strains were PG (phosphatidylglycerol), PGP-Me (phosphatidylglycerol phosphate methyl ester), PGS (phosphatidylglycerol sulfate) and five glycolipids chromatographically identical to S-TGD-1 (sulfated galactosyl mannosyl glucosyl diether), S-DGD-1 (sulfated mannosyl glucosyl diether), TGD-1 (galactosyl mannosyl glucosyl diether), DGD-1 (mannosyl glucosyl diether) and DGD-2 (an unknown diglycosyl diether). Phylogenetic analysis revealed that TBN4^T and strain TBN5 formed a distinct clade with genus Haladaptatus (showing 90.0–90.9% 16S rRNA gene similarities). The DNA G + C content of strain TBN4^T and strain TBN5 are 66.1 and 65.4 mol%, respectively. The DNA–DNA hybridization value between strain TBN4^T and strain TBN5 was 94.3%. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain TBN4^T and strain TBN5 represent a novel species in a new genus within the family Halobacteriaceae, for which the name Halorussus rarus gen. nov., sp. nov. is proposed. The type strain is TBN4^T (=CGMCC 1.10122^T = JCM 16429^T).     

<Emphasis Type="Italic">Ancylobacter abiegnus</Emphasis> sp. nov., an oligotrophic member of the xylotrophic mycobacterial community

A new species of the genus Ancylobacter, exemplified by strain Z-0056, was isolated from dystrophic humified waters formed by xylotrophic fungi grown on decaying spruce wood. The cells of strain Z-0056 (0.65–0.9 μm) are coccoid, gram-negative, with fimbriae, and nonmotile. The strain is pleomorphic and reproduces by nonuniform division. Strain Z-0056 is an aerobic organoheterotroph and a mesophilic and moderately acidophilic oligotrophic microorganism. As an inhabitant of dystrophic ultrafresh waters, strain Z-0056 is sensitive to NaCl. The bacterium utilizes organic acids (acetate, pyruvate, oxalate, gluconate, malate, succinate, and citrate), as well as xylose and xylan. The microorganism grows in a pH range of 4.0–8.0, with an optimum at pH 5.5. The temperature range for growth is 15–25°C, with an optimum at 20°C. According to its ecophysiological properties, strain Z-0056 belongs to the group of ombrophilic dissipotrophs. The DNA G+C base content is 66.8 mol %. According to phylogenetic analysis, strain Z-0056 belongs to the genus Ancylobacter. Strain Z-0056 showed the highest similarity (98.3%) with the type strain of the species A. oerskovii. The phenotypic and phylogenetic properties of strain Z-0056 support classification of this microorganism within the genus Ancylobacter as the novel species Ancylobacter abiegnus sp. nov.     

Cloning,expression and characterization of a halotolerant esterase from a marine bacterium <Emphasis Type="Italic">Pelagibacterium halotolerans</Emphasis> B2<Superscript>T</Superscript>

An esterase PE10 (279 aa) from Pelagibacterium halotolerans B2^T was cloned and overexpressed in Escherichia coli Rosetta in a soluble form. The deduced protein was 29.91 kDa and the phylogenetic analysis of the deduced amino acids sequence showed it represented a new family of lipolytic enzymes. The recombinant protein was purified by Ni–NTA affinity chromatography column and the characterization showed its optimal temperature and pH were 45 °C and pH 7.5, respectively. Substrate specificity study showed PE10 preferred short chain p-nitrophenyl esters and exhibited maximum activity toward p-nitrophenyl acetate. In addition, PE10 was a halotolerant esterase as it was still active under 4 M NaCl. Three-dimensional modeling of PE10 suggested that the high negative electrostatic potential on the surface may relevant to its tolerance to high salt environment. With this halotolerance property, PE10 could be a candidate for industrial use.     

Isolation and characterization of novel denitrifying alkalithermophiles, AT-1 and AT-2

Two novel denitrifying alkalithermophilic bacteria, AT-1 and AT-2, were isolated from manure-amended soil. The isolates grew at 35–65°C with an optimum temperature at 50–60°C, and pH 6.5–10.0 with an optimum pH at 9.5. Both isolates were Gram-positive, facultative anaerobic, non-motile rod-shaped bacteria. A phylogenetic analysis based on 16S rRNA sequence data indicated that both AT-1 and AT-2 are members of the genus Anoxybacillus. DNA-DNA hybridization revealed moderate relatedness between AT-1 and AT-2 and one phylogenetically related strain, A. pushchinensis K1 (69.5 and 69.1%, respectively). Comparative analysis of morphology and biochemical characteristics of the two isolates also showed similarity to A. pushchinensis K1. Based on these results, we identified AT-1 and AT-2 as A. pushchinensis. To our knowledge, this is the first report of denitrifying bacterium isolated from alkalithermophilic Anoxybacillus spp.     

Comparison of growth characteristics and roquefortin C production of<Emphasis Type="Italic">Penicillium roqueforti</Emphasis> from blue-veined cheese

The properties of 21 isolates ofPenicillium roqueforti from just as many commercial blue-veined cheeses, purchased from the Argentinean market (domestic and imported products) were comparatively examined. Isolates were investigated for their ability to grow at different temperatures, pH values and concentration of NaCl, as well as for their proteolytic and lipolytic activities, respectively. The potential of these strains to produce roquefortin in vitro, and the actual levels of roquefortin in 10 of these cheeses were analysed by TLC. All strains showed similar growth properties in aspects of salt concentration and pH-value of the medium, and all grew well at 10 °C. Only four strains showed proteolytic activity on casein agar, while all strains were lipolytic on trybutirin agar. After incubation at 25 °C for 16 days, all strains produced roquefortin in Yeast Extract Sucrose (25.6–426.7 μg/g) and in reconstituted (10%) sterile skim milk (26.9–488 μg/g). Roquefortin at >0.1 μg/g was also found in 9 out of 10 analysed samples of blue-veined cheeses (8 from Argentine, 1 from Spain), with a maximum value 3.6 μg/g. During the ripening process of blueveined cheese, production of roquefortin seems to be unavoidable. Care should be taken to select strains with low toxin production characteristics, to minimize potential health risks. Roquefortin C production byP. roqueforti in vitro was not correlated with roquefortin C levels found in cheese. Financial support: Research grants from the National University of Quilmes, Argentina     

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).     

Bacillus arsenicoselenatis, sp. nov., and Bacillus selenitireducens, sp. nov.: two haloalkaliphiles from Mono Lake, California that respire oxyanions of selenium and arsenic

Two gram-positive anaerobic bacteria (strains E1H and MLS10) were isolated from the anoxic muds of Mono Lake, California, an alkaline, hypersaline, arsenic-rich water body. Both grew by dissimilatory reduction of As(V) to As(III) with the concomitant oxidation of lactate to acetate plus CO₂. Bacillus arsenicoselenatis (strain E1H) is a spore-forming rod that also grew by dissimilatory reduction of Se(VI) to Se(IV). Bacillus selenitireducens (strain MLS10) is a short, non-spore-forming rod that grew by dissimilatory reduction of Se(IV) to Se(0). When the two isolates were cocultured, a complete reduction of Se(VI) to Se(0) was achieved. Both isolates are alkaliphiles and had optimal specific growth rates in the pH range of 8.5–10. Strain E1H had a salinity optimum at 60 g l^–1 NaCl, while strain MLS10 had optimal growth at lower salinities (24–60 g l^–1 NaCl). Both strains have limited abilities to grow with electron donors and acceptors other than those given above. Strain MLS10 demonstrated weak growth as a microaerophile and was also capable of fermentative growth on glucose, while strain E1H is a strict anaerobe. Comparative 16S rRNA gene sequence analysis placed the two isolates with other Bacillus spp. in the low G+C gram-positive group of bacteria. Received: 21 May 1998 / Accepted: 31 August 1998     

An unusual cyanobacterium from saline thermal waters with relatives from unexpected habitats

Cyanobacteria that grow above seawater salinity at temperatures above 45°C have rarely been studied. Cyanobacteria of this type of thermo-halophilic extremophile were isolated from siliceous crusts at 40–45°C in a geothermal seawater lagoon in southwest Iceland. Iceland Clone 2e, a Leptolyngbya morphotype, was selected for further study. This culture grew only at 45–50°C, in medium ranging from 28 to 94 g L⁻¹ TDS, It showed 3 doublings 24 h⁻¹ under continuous illumination. This rate at 54°C was somewhat reduced, and death occurred at 58°C. A comparison of the 16S rDNA sequence with all others in the NCBI database revealed 2 related Leptolyngbya isolates from a Greenland hot spring (13–16 g L⁻¹ TDS). Three other similar sequences were from Leptolyngbya isolates from dry, endolithic habitats in Yellowstone National Park. All 6 formed a phylogenetic clade, suggesting common ancestry. These strains shared many similarities to Iceland Clone 2e with respect to temperature and salinity ranges and optima. Two endolithic Leptolyngbya isolates, grown previously at 23°C in freshwater medium, grew well at 50°C but only in saline medium. This study shows that limited genotypic similarity may reveal some salient phenotypic similarities, even when the related cyanobacteria are from vastly different and remote habitats.     

Catalytic properties of mycelium-bound lipases from <Emphasis Type="Italic">Aspergillus niger</Emphasis> MYA 135

A constitutive level of a mycelium-bound lipolytic activity from Aspergillus niger MYA 135 was strongly increased by 97% in medium supplemented with 2% olive oil. The constitutive lipase showed an optimal activity in the pH range of 3.0–6.5, while the mycelium-bound lipase activity produced in the presence of olive oil had two pH optima at pH 4 and 7. Interestingly, both lipolytic sources were cold-active showing high catalytic activities in the temperature range of 4–8°C. These mycelium-bound lipase activities were also very stable in reaction mixtures containing methanol and ethanol. In fact, the constitutive lipase maintained almost 100% of its activity after exposure by 1 h at 37°C in ethanol. A simple methodology to evaluate suitable transesterification activities in organic solvents was also reported.     

Biodegradation of crude oil and pure hydrocarbons by extreme halophilic archaea from hypersaline coasts of the Arabian Gulf

Two extreme halophilic Haloferax strains and one strain each of Halobacterium and Halococcus were isolated from a hypersaline coastal area of the Arabian Gulf on a mineral salt medium with crude oil vapor as a sole source of carbon and energy. These archaea needed at least 1 M NaCl for growth in culture, and grew best in the presence of 4 M NaCl or more. Optimum growth temperatures lied between 40 and 45oC. The four archaea were resistant to the antibiotics chloramphenicol, cycloheximide, nalidixic acid, penicillin, streptomycin and tetracycline. The strains could grow on a wide scope of aliphatic and aromatic (both mono-and polynuclear) hydrocarbons, as sole sources of carbon and energy. Quantitative measurements revealed that these extreme halophilic prokaryotes could biodegrade crude oil (13–47%, depending on the strain and medium salinity), n-octadecane (28–67%) and phenanthrene (13–30%) in culture after 3 weeks of incubation. The rates of biodegradation by all strains were enhanced with increasing NaCl concentration in the medium. Optimal concentration was 3 M NaCl, but even with 4 M NaCl the hydrocarbon-biodegradation rates were higher than with 1 and 2 M NaCl. It was concluded that these archaea could contribute to self-cleaning and bioremediation of oil-polluted hypersaline environments.     

Molecular cloning and characterization of a new cold-active esterase from a deep-sea metagenomic library

A clone which conferred lipolytic activity at low temperature was identified from a fosmid library constructed from a South China Sea marine sediment sample. The gene responsible, estF, consisted of 1,080 bp that encoded 359 amino acid residues, with a typical N-terminal signal peptide of 28 amino acid residues. A phylogenetic analysis of amino acid sequence with other lipolytic enzymes revealed that EstF and seven closely related putative lipolytic enzymes comprised a unique clade in the phylogenetic tree. Moreover, these hypothetic esterases showed unique conservative sites in the amino acid sequence. The recombinant EstF was overexpressed and purified, and its biochemical properties were partially characterized. The optimal substrate for EstF to hydrolyze among a panel of p-nitrophenyl esters (C2 to C16) was p-nitrophenyl butyrate (C4), with a K _m of 0.46 mM. Activity quickly decreased with substrates containing an acyl chain length longer than 10 carbons. We found that EstF was active in the temperature range of 0–60°C, showed the best activity at 50°C, but was unstable at 60°C. It exhibited a high level of activity in the pH range of 7.0–10.0 showing the highest activity at pH 9.0.     

Biochemical and phylogenetic characterization of a novel terrestrial hyperthermophilic archaeon pertaining to the genus <Emphasis Type="Italic">Pyrococcus</Emphasis> from an Algerian hydrothermal hot spring

A hyperthermophilic anaerobic archeon, strain HT3, was isolated from hydrothermal hot spring in Northeast Algeria. The strain is a regular coccus, highly motile, obligatory anaerobic, heterotrophic. It utilizes proteinaceous complex media (peptone, tryptone or yeast extract). Sulfur is reduced to Hydrogen sulfide and enhances growth. It shares with other Pyrococcus species the heterotrophic mode of nutrition, the hyperthermophily, the ability to utilize amino acids as sole carbon and nitrogen sources and the ether lipid composition. The optimal growth occurs at 80–85°C, pH 7.5 and 1.5% NaCl. The G + C content was 43 mol%. Considering its morphology, physiological properties, nutritional features and phylogenetic analyses based on 16S rRNA gene sequencing, this strain is described as a new terrestrial isolate pertaining to the genus Pyrococcus.     

Identification of a new subfamily of salt-tolerant esterases from a metagenomic library of tidal flat sediment

To search for novel lipolytic enzymes, a metagenomic library was constructed from the tidal flat sediment of Ganghwa Island in South Korea. By functional screening using tributyrin agar plates, 3 clones were selected from among the 80,050 clones of the fosmid library. The sequence analysis revealed that those clones contained different open reading frames, which showed 50–57% amino acid identity with putative lipolytic enzymes in the database. Based on the phylogenetic analysis, they were identified to encode novel members, which form a distinct and new subfamily in the family IV of bacterial lipolytic enzymes. The consensus sequence, GT(S)SA(G)G, encompassing the active site serine of the enzymes was different from the GDSAG motif, conserved in the other subfamily. The genes were expressed in Escherichia coli and recombinant proteins were purified as active soluble forms. The enzymes showed the highest activity toward p-nitrophenyl valerate (C5) and exhibited optimum activities at mesophilic temperature ranges and slightly alkaline pH. In particular, the enzymes displayed salt tolerance with over 50% of the maximum activity remained in the presence of 3 M NaCl (or KCl). In this study, we demonstrated that the metagenomic approach using marine tidal flat sediment as a DNA source expanded the diversity of lipolytic enzyme-encoding genes.     

<Emphasis Type="Italic">Tropicibacter phthalicus</Emphasis> sp. nov., A Phthalate-Degrading Bacterium from Seawater

An aerobic, Gram-negative bacterial strain, designated KU27E1^T, which degrades phthalate and dimethylphthalate, was isolated from seawater obtained from the coastal region of Ishigaki Island, Japan. Cells are motile rods with polar flagella. Strain KU27E1^T grew at 15–30°C, pH 6.0–8.0, in the presence of 1.0–2.0% (w/v) NaCl. The 16S rRNA gene sequence analysis revealed that this strain was affiliated with the family Rhodobacteraceae in the class Alphaproteobacteria, and was most closely related to Tropicibacter naphthalenivorans (96.8%). The predominant respiratory lipoquinone was ubiquinone-10, and the major cellular fatty acid was C_18:1ω7c (88.5%). The G+C content of genomic DNA was 58.7 mol%. Based on the physiological, chemotaxonomic, and phylogenetic data, strain KU27E1^T is suggested to represent a novel species of the genus Tropicibacter, for which the name Tropicibacter phthalicus sp. nov. is proposed. The type strain of Tropicibacter phthalicus is designated as KU27E1^T (=JCM 17793^T = KCTC 23703^T).