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A Phytase-Based Reporter System for Identification of Functional Secretion Signals in Bifidobacteria.

Osswald A, Westermann C, Sun Z, Riedel CU - PLoS ONE (2015)

Bottom Line: Health-promoting effects have been attributed to a number of Bifidobacterium sp. strains.These effects as well as the ability to colonise the host depend on secreted proteins.Expression of a secreted cytosine deaminase led to a 100-fold reduced sensitivity of B. bifidum S17 to 5-fluorocytosine compared to the non-secreted cytosine deaminase suggesting efficient conversion of 5-fluorocytosine to the cytotoxic cancer drug 5-fluorouracil by cytosine deaminase occurred outside the bacterial cell.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Biotechnology, University of Ulm, 89068, Ulm, Germany.

ABSTRACT
Health-promoting effects have been attributed to a number of Bifidobacterium sp. strains. These effects as well as the ability to colonise the host depend on secreted proteins. Moreover, rational design of protein secretion systems bears the potential for the generation of novel probiotic bifidobacteria with improved health-promoting or therapeutic properties. To date, there is only very limited data on secretion signals of bifidobacteria available. Using in silico analysis, we demonstrate that all bifidobacteria encode the major components of Sec-dependent secretion machineries but only B. longum strains harbour Tat protein translocation systems. A reporter plasmid for secretion signals in bifidobacteria was established by fusing the coding sequence of the signal peptide of a sialidase of Bifidobacterium bifidum S17 to the phytase gene appA of E. coli. The recombinant strain showed increased phytase activity in spent culture supernatants and reduced phytase levels in crude extracts compared to the control indicating efficient phytase secretion. The reporter plasmid was used to screen seven predicted signal peptides in B. bifidum S17 and B. longum E18. The tested signal peptides differed substantially in their efficacy to mediate protein secretion in different host strains. An efficient signal peptide was used for expression and secretion of a therapeutically relevant protein in B. bifidum S17. Expression of a secreted cytosine deaminase led to a 100-fold reduced sensitivity of B. bifidum S17 to 5-fluorocytosine compared to the non-secreted cytosine deaminase suggesting efficient conversion of 5-fluorocytosine to the cytotoxic cancer drug 5-fluorouracil by cytosine deaminase occurred outside the bacterial cell. Selection of appropriate signal peptides for defined protein secretion might improve therapeutic efficacy as well as probiotic properties of bifidobacteria.

No MeSH data available.


Related in: MedlinePlus

Ca-phytate degradation of recombinant bifidobacteria expressing phytase with different signal peptides.Calcium phytate degradation by recombinant strains of B. bifidum S17 (A) and B. longum E18 (B) harbouring pMgapP-derived plasmids containing different SPs (S0-S6). The control plasmid pMgapP contains no SP and serves as a background control for expression of a non-secreted phytase. Overnight cultures of all strains were spotted in triplicate on RCM agar supplemented with 0.15% calcium phytate and imaged after anaerobic incubation for 48 h at 37°C. One representative spot of three independent cultures is shown.
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pone.0128802.g002: Ca-phytate degradation of recombinant bifidobacteria expressing phytase with different signal peptides.Calcium phytate degradation by recombinant strains of B. bifidum S17 (A) and B. longum E18 (B) harbouring pMgapP-derived plasmids containing different SPs (S0-S6). The control plasmid pMgapP contains no SP and serves as a background control for expression of a non-secreted phytase. Overnight cultures of all strains were spotted in triplicate on RCM agar supplemented with 0.15% calcium phytate and imaged after anaerobic incubation for 48 h at 37°C. One representative spot of three independent cultures is shown.

Mentions: All recombinant strains were analysed for phytase secretion using a phenotypic assay based on the degradation of insoluble Ca-phytate in solid medium (Fig 2). Clear zones of Ca-phytate degradation were observed for B. bifidum S17 strains harbouring pMgapS0P, pMgapS1P, pMgapS3P, pMgapS4P, and pMgapS6. By contrast, strains harbouring plasmids pMgapS2P and pMgapS5P did not display Ca-phytate degradation above background levels (pMgapP). A similar pattern of Ca-phytate degradation was observed for B. longum E18 strains, however at somewhat lower levels.


A Phytase-Based Reporter System for Identification of Functional Secretion Signals in Bifidobacteria.

Osswald A, Westermann C, Sun Z, Riedel CU - PLoS ONE (2015)

Ca-phytate degradation of recombinant bifidobacteria expressing phytase with different signal peptides.Calcium phytate degradation by recombinant strains of B. bifidum S17 (A) and B. longum E18 (B) harbouring pMgapP-derived plasmids containing different SPs (S0-S6). The control plasmid pMgapP contains no SP and serves as a background control for expression of a non-secreted phytase. Overnight cultures of all strains were spotted in triplicate on RCM agar supplemented with 0.15% calcium phytate and imaged after anaerobic incubation for 48 h at 37°C. One representative spot of three independent cultures is shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4472781&req=5

pone.0128802.g002: Ca-phytate degradation of recombinant bifidobacteria expressing phytase with different signal peptides.Calcium phytate degradation by recombinant strains of B. bifidum S17 (A) and B. longum E18 (B) harbouring pMgapP-derived plasmids containing different SPs (S0-S6). The control plasmid pMgapP contains no SP and serves as a background control for expression of a non-secreted phytase. Overnight cultures of all strains were spotted in triplicate on RCM agar supplemented with 0.15% calcium phytate and imaged after anaerobic incubation for 48 h at 37°C. One representative spot of three independent cultures is shown.
Mentions: All recombinant strains were analysed for phytase secretion using a phenotypic assay based on the degradation of insoluble Ca-phytate in solid medium (Fig 2). Clear zones of Ca-phytate degradation were observed for B. bifidum S17 strains harbouring pMgapS0P, pMgapS1P, pMgapS3P, pMgapS4P, and pMgapS6. By contrast, strains harbouring plasmids pMgapS2P and pMgapS5P did not display Ca-phytate degradation above background levels (pMgapP). A similar pattern of Ca-phytate degradation was observed for B. longum E18 strains, however at somewhat lower levels.

Bottom Line: Health-promoting effects have been attributed to a number of Bifidobacterium sp. strains.These effects as well as the ability to colonise the host depend on secreted proteins.Expression of a secreted cytosine deaminase led to a 100-fold reduced sensitivity of B. bifidum S17 to 5-fluorocytosine compared to the non-secreted cytosine deaminase suggesting efficient conversion of 5-fluorocytosine to the cytotoxic cancer drug 5-fluorouracil by cytosine deaminase occurred outside the bacterial cell.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Biotechnology, University of Ulm, 89068, Ulm, Germany.

ABSTRACT
Health-promoting effects have been attributed to a number of Bifidobacterium sp. strains. These effects as well as the ability to colonise the host depend on secreted proteins. Moreover, rational design of protein secretion systems bears the potential for the generation of novel probiotic bifidobacteria with improved health-promoting or therapeutic properties. To date, there is only very limited data on secretion signals of bifidobacteria available. Using in silico analysis, we demonstrate that all bifidobacteria encode the major components of Sec-dependent secretion machineries but only B. longum strains harbour Tat protein translocation systems. A reporter plasmid for secretion signals in bifidobacteria was established by fusing the coding sequence of the signal peptide of a sialidase of Bifidobacterium bifidum S17 to the phytase gene appA of E. coli. The recombinant strain showed increased phytase activity in spent culture supernatants and reduced phytase levels in crude extracts compared to the control indicating efficient phytase secretion. The reporter plasmid was used to screen seven predicted signal peptides in B. bifidum S17 and B. longum E18. The tested signal peptides differed substantially in their efficacy to mediate protein secretion in different host strains. An efficient signal peptide was used for expression and secretion of a therapeutically relevant protein in B. bifidum S17. Expression of a secreted cytosine deaminase led to a 100-fold reduced sensitivity of B. bifidum S17 to 5-fluorocytosine compared to the non-secreted cytosine deaminase suggesting efficient conversion of 5-fluorocytosine to the cytotoxic cancer drug 5-fluorouracil by cytosine deaminase occurred outside the bacterial cell. Selection of appropriate signal peptides for defined protein secretion might improve therapeutic efficacy as well as probiotic properties of bifidobacteria.

No MeSH data available.


Related in: MedlinePlus