Dataset Information

Biochemical and kinetic characterisation of a novel xylooligosaccharide-upregulated GH43 ?-d-xylosidase/?-l-arabinofuranosidase (BXA43) from the probiotic Bifidobacterium animalis subsp. lactis BB-12.

ABSTRACT: The Bifidobacterium animalis subsp. lactis BB-12 gene BIF_00092, assigned to encode a ?-d-xylosidase (BXA43) of glycoside hydrolase family 43 (GH43), was cloned with a C-terminal His-tag and expressed in Escherichia coli. BXA43 was purified to homogeneity from the cell lysate and found to be a dual-specificity exo-hydrolase active on para-nitrophenyl-?-d-xylopyranoside (pNPX), para-nitrophenyl-?-L-arabinofuranoside (pNPA), ?-(1???4)-xylopyranosyl oligomers (XOS) of degree of polymerisation (DP) 2-4, and birchwood xylan. A phylogenetic tree of the 92 characterised GH43 enzymes displayed five distinct groups (I?-?V) showing specificity differences. BXA43 belonged to group IV and had an activity ratio for pNPA:pNPX of 1:25. BXA43 was stable below 40°C and at pH 4.0-8.0 and showed maximum activity at pH 5.5 and 50°C. Km and kcat for pNPX were 15.6?±?4.2 mM and 60.6?±?10.8 s-1, respectively, and substrate inhibition became apparent above 18 mM pNPX. Similar kinetic parameters and catalytic efficiency values were reported for ?-d-xylosidase (XynB3) from Geobacillus stearothermophilus T?6 also belonging to group IV. The activity of BXA43 for xylooligosaccharides increased with the size and was 2.3 and 5.6 fold higher, respectively for xylobiose and xylotetraose compared to pNPX. BXA43 showed clearly metal inhibition for Zn2+ and Ag+, which is different to its close homologues. Multiple sequence alignment and homology modelling indicated that Arg505Tyr506 present in BXA43 are probably important for binding to xylotetraose at subsite +3 and occur only in GH43 from the Bifidobacterium genus.

SUBMITTER: Viborg AH

PROVIDER: S-EPMC3847938 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Biochemical and kinetic characterisation of a novel xylooligosaccharide-upregulated GH43 β-d-xylosidase/α-l-arabinofuranosidase (BXA43) from the probiotic Bifidobacterium animalis subsp. lactis BB-12.

Viborg Alexander Holm AH Sørensen Kim Ib KI Gilad Ofir O Steen-Jensen Daniel Bisgaard DB Dilokpimol Adiphol A Jacobsen Susanne S Svensson Birte B

AMB Express 20130911 1

The Bifidobacterium animalis subsp. lactis BB-12 gene BIF_00092, assigned to encode a β-d-xylosidase (BXA43) of glycoside hydrolase family 43 (GH43), was cloned with a C-terminal His-tag and expressed in Escherichia coli. BXA43 was purified to homogeneity from the cell lysate and found to be a dual-specificity exo-hydrolase active on para-nitrophenyl-β-d-xylopyranoside (pNPX), para-nitrophenyl-α-L-arabinofuranoside (pNPA), β-(1 → 4)-xylopyranosyl oligomers (XOS) of degree of polymerisation (DP) ...[more]

PMID: 24025736

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Project description:Commercial probiotic bacteria must be tested for acquired antibiotic resistance elements to avoid potential transfer to pathogens. The European Food Safety Authority recommends testing resistance using microdilution culture techniques previously used to establish inhibitory thresholds for the Bifidobacterium genus. Many Bifidobacterium animalis subsp. lactis strains exhibit increased resistance to tetracycline, historically attributed to the ribosomal protection gene tet(W). However, some strains that harbor genetically identical tet(W) genes show various inhibition levels, suggesting that other genetic elements also contribute to observed differences. Here, we adapted several molecular assays to confirm the inhibition of B. animalis subsp. lactis strains Bl-04 and HN019 and employed RNA sequencing to assess the transcriptional differences related to genomic polymorphisms. We detected specific stress responses to the antibiotic by correlating ATP concentration to number of viable genome copies from droplet digital PCR and found that the bacteria were still metabolically active in high drug concentrations. Transcriptional analyses revealed that several polymorphic regions, particularly a novel multidrug efflux transporter, were differentially expressed between the strains in each experimental condition, likely having phenotypic effects. We also found that the tet(W) gene was upregulated only during subinhibitory tetracycline concentrations, while two novel tetracycline resistance genes were upregulated at high concentrations. Furthermore, many genes involved in amino acid metabolism and transporter function were upregulated, while genes for complex carbohydrate utilization, protein metabolism, and clustered regularly interspaced short palindromic repeat(s) (CRISPR)-Cas systems were downregulated. These results provide high-throughput means for assessing antibiotic resistances of two highly related probiotic strains and determine the genetic network that contributes to the global tetracycline response.IMPORTANCEBifidobacterium animalis subsp. lactis is widely used in human food and dietary supplements. Although well documented to be safe, B. animalis subsp. lactis strains must not contain transferable antibiotic resistance elements. Many B. animalis subsp. lactis strains have different resistance measurements despite being genetically similar, and the reasons for this are not well understood. In the current study, we sought to examine how genomic differences between two closely related industrial B. animalis subsp. lactis strains contribute to different resistance levels. This will lead to a better understanding of resistance, identify future targets for analysis of transferability, and expand our understanding of tetracycline resistance in bacteria.

Dataset Information

Biochemical and kinetic characterisation of a novel xylooligosaccharide-upregulated GH43 ?-d-xylosidase/?-l-arabinofuranosidase (BXA43) from the probiotic Bifidobacterium animalis subsp. lactis BB-12.

Publications

Biochemical and kinetic characterisation of a novel xylooligosaccharide-upregulated GH43 β-d-xylosidase/α-l-arabinofuranosidase (BXA43) from the probiotic Bifidobacterium animalis subsp. lactis BB-12.

Similar Datasets

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