Dataset Information

Microbial diversity analysis and screening for novel xylanase enzymes from the sediment of the Lobios Hot Spring in Spain.

ABSTRACT: Here, we describe the metagenome composition of a microbial community in a hot spring sediment as well as a sequence-based and function-based screening of the metagenome for identification of novel xylanases. The sediment was collected from the Lobios Hot Spring located in the province of Ourense (Spain). Environmental DNA was extracted and sequenced using Illumina technology, and a total of 3.6 Gbp of clean paired reads was produced. A taxonomic classification that was obtained by comparison to the NCBI protein nr database revealed a dominance of Bacteria (93%), followed by Archaea (6%). The most abundant bacterial phylum was Acidobacteria (25%), while Thaumarchaeota (5%) was the main archaeal phylum. Reads were assembled into contigs. Open reading frames (ORFs) predicted on these contigs were searched by BLAST against the CAZy database to retrieve xylanase encoding ORFs. A metagenomic fosmid library of approximately 150,000 clones was constructed to identify functional genes encoding thermostable xylanase enzymes. Function-based screening revealed a novel xylanase-encoding gene (XynA3), which was successfully expressed in E. coli BL21. The resulting protein (41?kDa), a member of glycoside hydrolase family 11 was purified and biochemically characterized. The highest activity was measured at 80?°C and pH 6.5. The protein was extremely thermostable and showed 94% remaining activity after incubation at 60?°C for 24?h and over 70% remaining activity after incubation at 70?°C for 24?h. Xylanolytic activity of the XynA3 enzyme was stimulated in the presence of ?-mercaptoethanol, dithiothreitol and Fe3+ ions. HPLC analysis showed that XynA3 hydrolyzes xylan forming xylobiose with lower proportion of xylotriose and xylose. Specific activity of the enzyme was 9080 U/mg for oat arabinoxylan and 5080 U/mg for beechwood xylan, respectively, without cellulase activity.

SUBMITTER: Knapik K

PROVIDER: S-EPMC6671963 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Microbial diversity analysis and screening for novel xylanase enzymes from the sediment of the Lobios Hot Spring in Spain.

Knapik Kamila K Becerra Manuel M González-Siso María-Isabel MI

Scientific reports 20190801 1

Here, we describe the metagenome composition of a microbial community in a hot spring sediment as well as a sequence-based and function-based screening of the metagenome for identification of novel xylanases. The sediment was collected from the Lobios Hot Spring located in the province of Ourense (Spain). Environmental DNA was extracted and sequenced using Illumina technology, and a total of 3.6 Gbp of clean paired reads was produced. A taxonomic classification that was obtained by comparison to ...[more]

PMID: 31371784

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Project description:A fosmid library was constructed with the metagenomic DNA from the water of the Lobios hot spring (76°C, pH = 8.2) located in Ourense (Spain). Metagenomic sequencing of the fosmid library allowed the assembly of 9722 contigs ranging in size from 500 to 56,677 bp and spanning ~18 Mbp. 23,207 ORFs (Open Reading Frames) were predicted from the assembly. Biodiversity was explored by taxonomic classification and it revealed that bacteria were predominant, while the archaea were less abundant. The six most abundant bacterial phyla were Deinococcus-Thermus, Proteobacteria, Firmicutes, Acidobacteria, Aquificae, and Chloroflexi. Within the archaeal superkingdom, the phylum Thaumarchaeota was predominant with the dominant species "Candidatus Caldiarchaeum subterraneum." Functional classification revealed the genes associated to one-carbon metabolism as the most abundant. Both taxonomic and functional classifications showed a mixture of different microbial metabolic patterns: aerobic and anaerobic, chemoorganotrophic and chemolithotrophic, autotrophic and heterotrophic. Remarkably, the presence of genes encoding enzymes with potential biotechnological interest, such as xylanases, galactosidases, proteases, and lipases, was also revealed in the metagenomic library. Functional screening of this library was subsequently done looking for genes encoding lipolytic enzymes. Six genes conferring lipolytic activity were identified and one was cloned and characterized. This gene was named LOB4Est and it was expressed in a yeast mesophilic host. LOB4Est codes for a novel esterase of family VIII, with sequence similarity to ?-lactamases, but with unusual wide substrate specificity. When the enzyme was purified from the mesophilic host it showed half-life of 1 h and 43 min at 50°C, and maximal activity at 40°C and pH 7.5 with p-nitrophenyl-laurate as substrate. Interestingly, the enzyme retained more than 80% of maximal activity in a broad range of pH from 6.5 to 8.

Project description:A culture-independent molecular phylogenetic survey was carried out for the bacterial community in Obsidian Pool (OP), a Yellowstone National Park hot spring previously shown to contain remarkable archaeal diversity (S. M. Barns, R. E. Fundyga, M. W. Jeffries, and N. R. Page, Proc. Natl. Acad. Sci. USA 91:1609-1613, 1994). Small-subunit rRNA genes (rDNA) were amplified directly from OP sediment DNA by PCR with universally conserved or Bacteria-specific rDNA primers and cloned. Unique rDNA types among > 300 clones were identified by restriction fragment length polymorphism, and 122 representative rDNA sequences were determined. These were found to represent 54 distinct bacterial sequence types or clusters (> or = 98% identity) of sequences. A majority (70%) of the sequence types were affiliated with 14 previously recognized bacterial divisions (main phyla; kingdoms); 30% were unaffiliated with recognized bacterial divisions. The unaffiliated sequence types (represented by 38 sequences) nominally comprise 12 novel, division level lineages termed candidate divisions. Several OP sequences were nearly identical to those of cultivated chemolithotrophic thermophiles, including the hydrogen-oxidizing Calderobacterium and the sulfate reducers Thermodesulfovibrio and Thermodesulfobacterium, or belonged to monophyletic assemblages recognized for a particular type of metabolism, such as the hydrogen-oxidizing Aquificales and the sulfate-reducing delta-Proteobacteria. The occurrence of such organisms is consistent with the chemical composition of OP (high in reduced iron and sulfur) and suggests a lithotrophic base for primary productivity in this hot spring, through hydrogen oxidation and sulfate reduction. Unexpectedly, no archaeal sequences were encountered in OP clone libraries made with universal primers. Hybridization analysis of amplified OP DNA with domain-specific probes confirmed that the analyzed community rDNA from OP sediment was predominantly bacterial. These results expand substantially our knowledge of the extent of bacterial diversity and call into question the commonly held notion that Archaea dominate hydrothermal environments. Finally, the currently known extent of division level bacterial phylogenetic diversity is collated and summarized.

Project description:A polyphasic study was designed to resolve the taxonomic position of isolate MGRD01-02T which was recovered from an acidic hot spring in Indonesia and assigned to the genus Actinospica. Phylogenetic analyses based on 16S rRNA gene sequences show that the isolate is most closely related to the type strains of Actinospica acidiphila (98.5%), Actinospica robiniae (97.8%) and Actinospica durhamensis (96.8%). Morphological and chemotaxonomic data underpin the assignment of the isolate to the genus Actinospica as it forms an extensively branched substrate mycelium which carries tufts of white aerial hyphae that differentiate into straight to flexuous chains of cylindrical spores with faint rugose surfaces, contains 2,6-diamino-3-hydroxydiaminopimelic acid in the peptidoglycan, mixtures of hydrogenated menaquinones with nine isoprene units, iso-C 15:O and iso-C 16:O as major fatty acids and phosphatidylethanolamine as the diagnostic phospholipid. Whole-genome sequence analyses show that the isolate, A. durhamensis CSCA 57T and Actinocrinis puniceicyclus DSM 45168T have genome sizes of 7.9, 9.6 and 6.7 Mbp, respectively. A phylogenomic tree shows that they form distinct branches in a well-supported clade, a result supported by associated phenotypic data. Average nucleotide identity and digital DNA:DNA hybridization similarities are below the recommended thresholds for assigning strains to the same species; they also indicate that isolate MGRD01-02T is most closely related to the A. durhamensis and A. robiniae strains. Corresponding amino acid identity and conserved protein data not only support these relationships but also confirm the taxonomic integrity of the genus Actinocrinis. Based on these results, it is proposed that isolate MGRD01-02T (= CCMM B1308T = ICEBB-09T = NCIMB 15218T) be classified in the genus Actinospica as Actinospica acidithermotolerans sp. nov. The draft genome of the isolate and its closest phylogenomic neighbours contain biosynthetic gene clusters with the potential to produce new natural products, notably antibiotics.

Dataset Information

Microbial diversity analysis and screening for novel xylanase enzymes from the sediment of the Lobios Hot Spring in Spain.

Publications

Microbial diversity analysis and screening for novel xylanase enzymes from the sediment of the Lobios Hot Spring in Spain.

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