LC-ESI-MS/MS identification of polar lipids of two thermophilic Anoxybacillus bacteria containing a unique lipid pattern.

Phospholipids and glycolipids from two recently described species belonging to the thermophilic genus Anoxybacillus were analyzed by liquid chromatography-electrospray tandem mass spectrometry (LC/ESI-MS/MS). Analysis of total lipids from the facultatively anaerobic A. bogrovensis on a HILIC (Hydrophilic Interaction LIquid Chromatography) column succeeded in separating diacyl- and plasmalogen phospholipids. The LC/ESI-MS/MS analysis of the strict aerobe A. rupiensis revealed the presence of different unique polar lipids, predominantly alanyl-, lysyl-, and glucosyl-phosphatidylglycerols and cardiolipins. Each of the classes of polar lipids was then analyzed by means of the ESI-MS/MS and more than 140 molecular species of six lipid classes from A. bogrovensis and nearly 200 molecular species of nine classes of polar lipids from A. rupiensis were identified. Five classes of unidentified polar lipids were detected in both strains. Plasmalogens were thus determined for the first time in a facultatively anaerobic bacterium, i.e. A. bogrovensis.

High Archaea diversity in Varvara hot spring, Bulgaria.

The phylogeny of the latest recognized domain, Archaea, is still complicated and it is largely based on environmental sequences. A culture independent molecular phylogenetic analysis revealed high Archaea diversity in a terrestrial hot spring, village Varvara, Bulgaria. A total of 35 archaeal operational taxonomic units (OTUs) belonging to three of the classified five Archaea phyla were identified. Most of the sequences were affiliated with the phylum Crenarchaeota (23), grouped in four branches. The rest of the sequences showed highest similarity to the unidentified archaeal clones (9), Euryarchaeota (2), and "Korarchaeota " (1). Eight (23%) of the sequenced 16S rDNAs didn't have known close relatives and represented new and diverse OTUs, four of them forming a new archaeal subgroup without close described sequences or culturable relatives. A sequence affiliated with "Korarchaeota " showed low similarity (90%) to the closest neighbor and both sequences formed unique branch in this phylum. Consequently, the constructed archaeal libraries are characterized by (1) high proportion of OTUs representing uncultivated archaeal phylogroups, (2) the abundance of novel phylotype sequences, (3) the presence of high proportions of Crenarchaeota phylotypes unrelated to cultivated organisms and (4) the presence of a sequence only distantly related to "Korarchaeota " phylum.

Physicochemical characteristics of a thermostable gellan lyase from Geobacillus stearothermophilus 98.

A purified thermostable gellan lyase, produced by a thermophilic bacterium, Geobacillus stearothermophilus 98, was characterized in relation to its physicochemical properties. The gellan lyase was established to have a molecular weight of 216 kDa, defined by capillary gel electrophoresis. Amino acid analysis revealed high quantities of Lys, His, Ala, Val, Ile, Glx, and Pro residues. The circular dichroism revealed 45% beta-structure and practically lack of a-spiral domains. Kinetic studies showed high affinity of the enzyme to gellan as a substrate (Km = 0.21 microM). The thermal denaturation investigated by cicular dichroism showed a highly cooperative transition with a midpoint (Tm) at about 75 degrees C. A single product was identified after enzyme action on gellan. Large exothermic aggregation near Tm was observed by differential scanning calorimetry. Two types of gellan lyase crystals were reproducibly isolated.

Anoxybacillus bogrovensis sp. nov., a novel thermophilic bacterium isolated from a hot spring in Dolni Bogrov, Bulgaria.

A novel moderately thermophilic bacterium, designated strain BT 13(T), was isolated from a geothermal water source in Dolni Bogrov, near Sofia, Bulgaria. The isolate was spore-forming, Gram-positive, facultatively anaerobic, alkalitolerant and heterotrophic, and was able to ferment a wide variety of carbon sources including d-glucose, sucrose, l-arabinose, l-rhamnose, starch, sorbitol and glycogen. Strain BT 13(T) grew optimally at pH 8.0 and 65 degrees C. Intracellular amylolytic activity was registered with glucose as the main product of starch hydrolysis. Phylogenetic analysis based on the 16S rRNA gene revealed that the strain belonged to the genus Anoxybacillus, the closest relatives being Anoxybacillus flavithermus and Anoxybacillus kamchatkensis. The DNA G+C content was 44.1 mol%. The fatty acid profile with a content of iso-branched fatty acids of around 80 % of the total fatty acids is similar to that of recognized Anoxybacillus species. On the basis of genotypic differentiation and significant differences in phenotypic characteristics, it was concluded that strain BT 13(T) represents a novel species of the genus Anoxybacillus, for which the name Anoxybacillus bogrovensis sp. nov. is proposed. The type strain is BT 13(T) (=DSM 17956(T)=NBIMCC 8427(T)).

Anoxybacillus rupiensis 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 degrees 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.

Biosynthesis of a thermostable gellan lyase by newly isolated and characterized strain of Geobacillus stearothermophilus 98.

The thermophilic strain able to degrade gellan was isolated from Bulgarian hot spring. According to its morphological and biochemical properties and by partial sequencing of its 16S rDNA, it was classified as Geobacillus stearothermophilus. It grew in a synthetic medium with gellan as the only carbon source with a specific growth rate of 0.69 h(-1) and generation time of 60 min. The strain produced thermostable gellan lyase extracellularly during exponential phase. Its synthesis was inducible; the enzyme was not registered in culture liquid without gellan. The enzyme activity was increased tenfold in conditions of continuous cultivation compared to data from batch fermentations and enzyme productivity was almost sixfold higher. The enzyme showed optimal activity at 75 degrees C in a very large pH area 4-8.5. This enzyme is the first reported thermostable gellan lyase, its residual activity was 100% after 24 h incubation at 60 degrees C and its half-life was 60 min at 70 degrees C.