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Genome-wide analysis of signal peptide functionality in Lactobacillus plantarum WCFS1.

Mathiesen G, Sveen A, Brurberg MB, Fredriksen L, Axelsson L, Eijsink VG - BMC Genomics (2009)

Bottom Line: The results reveal considerable variation between the SPs that is at least in part dependent on the protein that is secreted.The results for NucA provide some hints as to the sequence-based prediction of SP functionality, but the general conclusion is that such prediction is difficult.The vector library generated in this study is based on exchangeable cassettes and provides a powerful tool for rapid experimental screening of SPs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Norwegian University of Life Sciences, Center for Molecular Microbiology, Department of Chemistry Biotechnology and Food Science, Chr. M. Falsensvei 1, P.O. Box 5003, N-1432 As, Norway. geir.mathiesen@umb.no

ABSTRACT

Background: Lactobacillus plantarum is a normal, potentially probiotic, inhabitant of the human gastrointestinal (GI) tract. The bacterium has great potential as food-grade cell factory and for in situ delivery of biomolecules. Since protein secretion is important both for probiotic activity and in biotechnological applications, we have carried out a genome-wide experimental study of signal peptide (SP) functionality.

Results: We have constructed a library of 76 Sec-type signal peptides from L. plantarum WCFS1 that were predicted to be cleaved by signal peptidase I. SP functionality was studied using staphylococcal nuclease (NucA) as a reporter protein. 82% of the SPs gave significant extracellular NucA activity. Levels of secreted NucA varied by a dramatic 1800-fold and this variation was shown not to be the result of different mRNA levels. For the best-performing SPs all produced NucA was detected in the culture supernatant, but the secretion efficiency decreased for the less well performing SPs. Sequence analyses of the SPs and their cognate proteins revealed four properties that correlated positively with SP performance for NucA: high hydrophobicity, the presence of a transmembrane helix predicted by TMHMM, the absence of an anchoring motif in the cognate protein, and the length of the H+C domain. Analysis of a subset of SPs with a lactobacillal amylase (AmyA) showed large variation in production levels and secretion efficiencies. Importantly, there was no correlation between SP performance with NucA and the performance with AmyA.

Conclusion: This is the first comprehensive experimental study showing that predicted SPs in the L. plantarum genome actually are capable of driving protein secretion. The results reveal considerable variation between the SPs that is at least in part dependent on the protein that is secreted. Several SPs stand out as promising candidates for efficient secretion of heterologous proteins in L. plantarum. The results for NucA provide some hints as to the sequence-based prediction of SP functionality, but the general conclusion is that such prediction is difficult. The vector library generated in this study is based on exchangeable cassettes and provides a powerful tool for rapid experimental screening of SPs.

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Scanning electron microscopy images of recombinant L. plantarum WCFS1, harvested at ~OD600 1.7. A, non-induced cells harboring pLp_2940sAmy; B, induced cells harboring pLp_2940sAmy; C, induced cells harboring pLp_0297sAmy.
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Figure 4: Scanning electron microscopy images of recombinant L. plantarum WCFS1, harvested at ~OD600 1.7. A, non-induced cells harboring pLp_2940sAmy; B, induced cells harboring pLp_2940sAmy; C, induced cells harboring pLp_0297sAmy.

Mentions: Using silver stained SDS-PAGE gels (additional file 3), the secreted amylase could be detected in non-concentrated culture media and the relative intensities of the bands correlated well with the relative levels of enzyme activities. In the case of the pLp_2940sAmy construct, which leads to high total amylase levels, cell lysis was observed. Scanning electron microscopy showed that induced pLp_2940sAmy containing cells had an elongated shape that differed drastically from the shape of non-induced pLp_2940sAmy containing cells and induced cells containing other secretion constructs such as pLp_0297sAmy (Figure 4).


Genome-wide analysis of signal peptide functionality in Lactobacillus plantarum WCFS1.

Mathiesen G, Sveen A, Brurberg MB, Fredriksen L, Axelsson L, Eijsink VG - BMC Genomics (2009)

Scanning electron microscopy images of recombinant L. plantarum WCFS1, harvested at ~OD600 1.7. A, non-induced cells harboring pLp_2940sAmy; B, induced cells harboring pLp_2940sAmy; C, induced cells harboring pLp_0297sAmy.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2748100&req=5

Figure 4: Scanning electron microscopy images of recombinant L. plantarum WCFS1, harvested at ~OD600 1.7. A, non-induced cells harboring pLp_2940sAmy; B, induced cells harboring pLp_2940sAmy; C, induced cells harboring pLp_0297sAmy.
Mentions: Using silver stained SDS-PAGE gels (additional file 3), the secreted amylase could be detected in non-concentrated culture media and the relative intensities of the bands correlated well with the relative levels of enzyme activities. In the case of the pLp_2940sAmy construct, which leads to high total amylase levels, cell lysis was observed. Scanning electron microscopy showed that induced pLp_2940sAmy containing cells had an elongated shape that differed drastically from the shape of non-induced pLp_2940sAmy containing cells and induced cells containing other secretion constructs such as pLp_0297sAmy (Figure 4).

Bottom Line: The results reveal considerable variation between the SPs that is at least in part dependent on the protein that is secreted.The results for NucA provide some hints as to the sequence-based prediction of SP functionality, but the general conclusion is that such prediction is difficult.The vector library generated in this study is based on exchangeable cassettes and provides a powerful tool for rapid experimental screening of SPs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Norwegian University of Life Sciences, Center for Molecular Microbiology, Department of Chemistry Biotechnology and Food Science, Chr. M. Falsensvei 1, P.O. Box 5003, N-1432 As, Norway. geir.mathiesen@umb.no

ABSTRACT

Background: Lactobacillus plantarum is a normal, potentially probiotic, inhabitant of the human gastrointestinal (GI) tract. The bacterium has great potential as food-grade cell factory and for in situ delivery of biomolecules. Since protein secretion is important both for probiotic activity and in biotechnological applications, we have carried out a genome-wide experimental study of signal peptide (SP) functionality.

Results: We have constructed a library of 76 Sec-type signal peptides from L. plantarum WCFS1 that were predicted to be cleaved by signal peptidase I. SP functionality was studied using staphylococcal nuclease (NucA) as a reporter protein. 82% of the SPs gave significant extracellular NucA activity. Levels of secreted NucA varied by a dramatic 1800-fold and this variation was shown not to be the result of different mRNA levels. For the best-performing SPs all produced NucA was detected in the culture supernatant, but the secretion efficiency decreased for the less well performing SPs. Sequence analyses of the SPs and their cognate proteins revealed four properties that correlated positively with SP performance for NucA: high hydrophobicity, the presence of a transmembrane helix predicted by TMHMM, the absence of an anchoring motif in the cognate protein, and the length of the H+C domain. Analysis of a subset of SPs with a lactobacillal amylase (AmyA) showed large variation in production levels and secretion efficiencies. Importantly, there was no correlation between SP performance with NucA and the performance with AmyA.

Conclusion: This is the first comprehensive experimental study showing that predicted SPs in the L. plantarum genome actually are capable of driving protein secretion. The results reveal considerable variation between the SPs that is at least in part dependent on the protein that is secreted. Several SPs stand out as promising candidates for efficient secretion of heterologous proteins in L. plantarum. The results for NucA provide some hints as to the sequence-based prediction of SP functionality, but the general conclusion is that such prediction is difficult. The vector library generated in this study is based on exchangeable cassettes and provides a powerful tool for rapid experimental screening of SPs.

Show MeSH
Related in: MedlinePlus