Galactomannan Catabolism Conferred by a Polysaccharide Utilization Locus of Bacteroides ovatus: ENZYME SYNERGY AND CRYSTAL STRUCTURE OF A ß-MANNANASE.

A recently identified polysaccharide utilization locus (PUL) from Bacteroides ovatus ATCC 8483 is transcriptionally up-regulated during growth on galacto- and glucomannans. It encodes two glycoside hydrolase family 26 (GH26) ß-mannanases, BoMan26A and BoMan26B, and a GH36 α-galactosidase, BoGal36A. The PUL also includes two glycan-binding proteins, confirmed by ß-mannan affinity electrophoresis. When this PUL was deleted, B. ovatus was no longer able to grow on locust bean galactomannan. BoMan26A primarily formed mannobiose from mannan polysaccharides. BoMan26B had higher activity on galactomannan with a high degree of galactosyl substitution and was shown to be endo-acting generating a more diverse mixture of oligosaccharides, including mannobiose. Of the two ß-mannanases, only BoMan26B hydrolyzed galactoglucomannan. A crystal structure of BoMan26A revealed a similar structure to the exo-mannobiohydrolase CjMan26C from Cellvibrio japonicus, with a conserved glycone region (-1 and -2 subsites), including a conserved loop closing the active site beyond subsite -2. Analysis of cellular location by immunolabeling and fluorescence microscopy suggests that BoMan26B is surface-exposed and associated with the outer membrane, although BoMan26A and BoGal36A are likely periplasmic. In light of the cellular location and the biochemical properties of the two characterized ß-mannanases, we propose a scheme of sequential action by the glycoside hydrolases encoded by the ß-mannan PUL and involved in the ß-mannan utilization pathway in B. ovatus. The outer membrane-associated BoMan26B initially acts on the polysaccharide galactomannan, producing comparably large oligosaccharide fragments. Galactomanno-oligosaccharides are further processed in the periplasm, degalactosylated by BoGal36A, and subsequently hydrolyzed into mainly mannobiose by the ß-mannanase BoMan26A.

A ß-mannan utilization locus in Bacteroides ovatus involves a GH36 α-galactosidase active on galactomannans.

The Bacova_02091 gene in the ß-mannan utilization locus of Bacteroides ovatus encodes a family GH36 α-galactosidase (BoGal36A), transcriptionally upregulated during growth on galactomannan. Characterization of recombinant BoGal36A reveals unique properties compared to other GH36 α-galactosidases, which preferentially hydrolyse terminal α-galactose in raffinose family oligosaccharides. BoGal36A prefers hydrolysing internal galactose substitutions from intact and depolymerized galactomannan. BoGal36A efficiently releases (> 90%) galactose from guar and locust bean galactomannans, resulting in precipitation of the polysaccharides. As compared to other GH36 structures, the BoGal36A 3D model displays a loop deletion, resulting in a wider active site cleft which likely can accommodate a galactose-substituted polymannose backbone.

Paenibacillus marinum sp. nov., a thermophilic xylanolytic bacterium isolated from a marine hot spring in Tunisia.

Among a large collection of Tunisian hot springs bacterial isolates a bacterial strain, THE22(T) , with xylanolytic properties was identified. The bacterium was isolated from a natural hot spring "Ain Echefa" at Mediteranean sea (Korbous, North-Eastern Tunisia). The novel strain was Gram positive, spore-forming, rod-shaped, facultatively anaerobic and grew optimally under conditions of 55 °C, 1% (w/v) NaCl and pH 7-8. The 16S rRNA gene sequence analysis showed that strain THE22(T) fell within the radiation of the cluster comprising Paenibacillus species with Paenibacillus phyllosphaerae PALXIL04(T) as the closest phylogenetic neighbour (95.8%). The predominant components in the fatty methyl ester profile were iso-C16:0 (34.46%), C16:0 (19.64%), anteiso-C15:0 (19.18%) and anteiso-C17:0 (18.11%). The major respiratory quinone was menaquinone-7 (MK-7). The diamino acid found in the cell-wall peptidoglycan was meso-diaminopimelic acid. The base composition of DNA was 56 mol%. Based on the polyphasic taxonomic data, strain THE-22(T) (=DSM 18499(T) = LMG 23758(T) ) was recognized as a novel species within the genus Paenibacillus. The name Paenibacillus marinum sp. nov. is proposed.

Characterization of Deinococcus sahariens sp. nov., a radiation-resistant bacterium isolated from a Saharan hot spring.

An ultraviolet-radiation-resistant, Gram-positive, orange-pigmented, thermophilic and strictly aerobic cocci was isolated from Saharan water hot spring in Tunisia. The newly isolated bacterium, designated HAN-23(T), was identified based on polyphasic taxonomy including genotypic, phenotypic and chemotaxonomic characterization. Phylogenetic analysis based on 16S rRNA gene sequences placed this strain within Deinococcus genus. However, strain HAN-23(T) is different from recognized species of the genus Deinococcus, showing less than 94.0% similarity values to its closest relatives. The predominant cellular fatty acids determined by gas chromatography were iso-C(15:0), iso-C(17:0) and iso C(17:1) ω9c. The major respiratory quinone was MK-8. The DNA G + C content was 66.9 mol%. DNA-DNA hybridization measurements revealed low DNA relatedness (6%) between the novel isolate and its closest neighbor, the type strain Deinococcus geothermalis DSM 11300. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, strain HAN-23(T) represents a novel species of the genus Deinococcus, for which the name Deinococcus sahariens sp. nov. is proposed, the type strain being HAN-23(T) (=DSM 18496(T); LMG 23756(T)).

Caldimonas hydrothermale sp. nov., a novel thermophilic bacterium isolated from Roman hot bath in south Tunisia.

A polyphasic approach was used to characterize a bacterium, HAN-85(T), isolated from thermal water in natural thermal spring at Tozeur, an oasis in southwest Tunisia. The novel isolate was thermophilic, strictly aerobic and amylolytic bacterium, which stained Gram negative. Cells were short rods motile by means of a single polar flagellum. Their optimum temperature and pH required for growth were 55 degrees C and pH 7, respectively. Comparative 16S rRNA gene sequence analyses showed that strain HAN-85(T) belonged to the genus Caldimonas, with highest sequence similarity to the type strains Caldimonas manganoxidans and Caldimonas taiwanensis. DNA-DNA hybridization measurements revealed low DNA relatedness (35.2-44.5%) between the novel isolate and its closest relative, C. manganoxidans. The major cellular fatty acid components were 16:0, 17:0 cyclo and summed feature 3. The DNA G+C content was 68.3 mol%. Taken together, the results of DNA-DNA hybridization, fatty acids profile, physiological tests and biochemical analyses have allowed the genotypic and phenotypic differentiation of the isolate from currently recognized Caldimonas species. Therefore, we suggest that this isolate is a novel species within the genus Caldimonas and propose that it should be named Caldimonas hydrothermale sp. nov. The type strain is HAN-85(T) (=DSM 18497(T) =LMG 23755(T)).