Functional screening of a human saliva metagenomic DNA reveal novel resistance genes against sodium hypochlorite and chlorhexidine

Objective Many sections of the health care system are facing a major challenge making infectious disease problematic to treat; antimicrobial resistance (AMR). Identification and surveillance of the resistome have been highlighted as one of the strategies to overcome the problem. This study aimed to screen for AMR genes in an oral microbiota, a complex microbial system continuously exposed to antimicrobial agents commonly used in dental practice. Materials and methods As a significant part of the oral microbiome cannot be conventionally cultured, a functional metagenomic approach was chosen. The human oral metagenomic DNA was extracted from saliva samples collected from 50 healthy volunteers in Norway. The oral metagenomic library was then constructed by ligating partially digested oral metagenome into pSMART BAC vector and introducing into Escherichia coli. The library was screened against antimicrobials in dental practices. All resistant clones were selected and analyzed. Results Screening of the oral metagenomic library against different antimicrobials detected multiple clones with resistance against chlorhexidine, triclosan, erythromycin, tetracycline, and sodium hypochlorite. Bioinformatic analysis revealed both already known resistance genes, including msr, mef(A), tetAB(46), and fabK, and genes that were not previously described to confer resistance, including recA and accB conferring resistance to sodium hypochlorite and chlorhexidine, respectively. Conclusion Multiple clones conferring resistance to antimicrobials commonly used in dental practices were detected, containing known and novel resistant genes by functional-based metagenomics. There is a need for more studies to increase our knowledge in the field.

Biochemical characterization of a family IV esterase with R-form enantioselectivity from a compost metagenomic library

A novel family IV esterase (hormone-sensitive lipase, HSL) gene, est15L, was isolated from a compost metagenomic library. Encoded Est15L comprised 328 amino acids with a molecular weight of 34,770 kDa and was an intracellular esterase without a signal peptide. The multiple sequence alignment (MSA) of Est15L with other family IV esterases showed conserved regions such as HGG, DYR, GXSXG, DPL, and GXIH. Native Est15L was a dimeric form from the results of size exclusion chromatography. It was optimally active at 50 ℃ and pH 9.0, indicating alkaline esterase. However, it showed a low thermostability with half-lives of 30.3 at 30 ℃ and 2.7 min at 40 ℃. It preferred p-nitrophenyl butyrate (C4) with Km and Vmax values of 0.28 mM and 270.8 U/mg, respectively. Est15L was inhibited by organic solvents such as 30% methanol, isopropanol, and acetonitrile with residual activities of 12.5, 0.9, and 0.3%, respectively. It was also inhibited by 1% SDS and 1% PMSF; however, Est15L maintained its activity at 1% Triton X-100 and EDTA. Est15L was inhibited by Cu2+, Zn2+, Mn2+, Co2+, Fe2+, and Na+. In addition, Est15L hydrolyzed glyceryl tributyrate with a residual substrate amount of 43.7% at 60 min but could not hydrolyze the oils (fish and olive) and glyceryl trioleate. Interestingly, Est15L showed significant enantioselectivity toward the R-form with a residual substrate amount of 44.6%, lower than that of the S-form (83.5%). Considering its properties, Est15L can be a potential candidate for chemical reactions, such as the synthesis of pharmaceutical compounds.

Characterization of a highly xylose tolerant β-xylosidase isolated from high temperature horse manure compost

Background There is a continued need for improved enzymes for industry. β-xylosidases are enzymes employed in a variety of industries and although many wild-type and engineered variants have been described, enzymes that are highly tolerant of the products produced by catalysis are not readily available and the fundamental mechanisms of tolerance are not well understood. Results Screening of a metagenomic library constructed of mDNA isolated from horse manure compost for β-xylosidase activity identified 26 positive hits. The fosmid clones were sequenced and bioinformatic analysis performed to identity putative β-xylosidases. Based on the novelty of its amino acid sequence and potential thermostability one enzyme (XylP81) was selected for expression and further characterization. XylP81 belongs to the family 39 β-xylosidases, a comparatively rarely found and characterized GH family. The enzyme displayed biochemical characteristics (KM—5.3 mM; Vmax—122 U/mg; kcat—107; Topt—50 °C; pHopt—6) comparable to previously characterized glycoside hydrolase family 39 (GH39) β-xylosidases and despite nucleotide identity to thermophilic species, the enzyme displayed only moderate thermostability with a half-life of 32 min at 60 °C. Apart from acting on substrates predicted for β-xylosidase (xylobiose and 4-nitrophenyl-β-D-xylopyranoside) the enzyme also displayed measurable α-L-arabainofuranosidase, β-galactosidase and β-glucosidase activity. A remarkable feature of this enzyme is its ability to tolerate high concentrations of xylose with a Ki of 1.33 M, a feature that is highly desirable for commercial applications. Conclusions Here we describe a novel β-xylosidase from a poorly studied glycosyl hydrolase family (GH39) which despite having overall kinetic properties similar to other bacterial GH39 β-xylosidases, displays unusually high product tolerance. This trait is shared with only one other member of the GH39 family, the recently described β-xylosidases from Dictyoglomus thermophilum. This feature should allow its use as starting material for engineering of an enzyme that may prove useful to industry and should assist in the fundamental understanding of the mechanism by which glycosyl hydrolases evolve product tolerance.

Multifunctional alkalophilic α-amylase with diverse raw seaweed degrading activities

Uncultured microbes are an important resource for the discovery of novel enzymes. In this study, an amylase gene (amy2587) that codes a protein with 587 amino acids (Amy2587) was obtained from the metagenomic library of macroalgae-associated bacteria. Recombinant Amy2587 was expressed in Escherichia coli BL21 (DE3) and was found to simultaneously possess α-amylase, agarase, carrageenase, cellulase, and alginate lyase activities. Moreover, recombinant Amy2587 showed high thermostability and alkali resistance which are important characteristics for industrial application. To investigate the multifunctional mechanism of Amy2587, three motifs (functional domains) in the Amy2587 sequence were deleted to generate three truncated Amy2587 variants. The results showed that, even though these functional domains affected the multiple substrates degrading activity of Amy2587, they did not wholly explain its multifunctional characteristics. To apply the multifunctional activity of Amy2587, three seaweed substrates (Grateloupia filicina, Chondrus ocellatus, and Scagassum) were digested using Amy2587. After 2 h, 6 h, and 24 h of digestion, 121.2 ± 4 µg/ml, 134.8 ± 6 µg/ml, and 70.3 ± 3.5 µg/ml of reducing sugars were released, respectively. These results show that Amy2587 directly and effectively degraded three kinds of raw seaweeds. This finding provides a theoretical basis for one-step enzymatic digestion of raw seaweeds to obtain seaweed oligosaccharides.

Evaluation of the Effects of Library Preparation Procedure and Sample Characteristics on the Accuracy of Metagenomic Profiles

Metagenomic library preparation methods and sequencing technologies continue to advance rapidly, allowing researchers to characterize microbial communities in previously underexplored environmental samples and systems. However, widely accepted standardized library preparation methods can be cost-prohibitive.