Heterologous Protein Secretion in Lactobacilli with Modified pSIP Vectors

<div><p>We describe new variants of the modular pSIP-vectors for inducible gene expression and protein secretion in lactobacilli. The basic functionality of the pSIP system was tested in <i>Lactobacillus</i> strains representing 14 species using pSIP411, which harbors the broad-host-range <i>Lactococcus lactis</i> SH71_rep replicon and a β-glucuronidase encoding reporter gene. In 10 species, the inducible gene expression system was functional. Based on these results, three pSIP vectors with different signal peptides were modified by replacing their narrow-host-range <i>L. plantarum</i> 256_rep replicon with SH71_rep and transformed into strains of five different species of <i>Lactobacillus</i>. All recombinant strains secreted the target protein NucA, albeit with varying production levels and secretion efficiencies. The Lp_3050 derived signal peptide generally resulted in the highest levels of secreted NucA. These modified pSIP vectors are useful tools for engineering a wide variety of <i>Lactobacillus</i> species.</p></div

Silver stained SDS-PAGE gel showing NucA in cell free supernatants from <i>Lactobacillus plantarum</i> WCFS1 harboring different expression vectors.

<p>The vectors differ with respect to the signal peptide (Lp_0373, Lp_3050 or Lp_2578) and the replicon (256_rep or SH71_rep), as indicated in the Figure. The sample size was 15 µl (Lp_3050 and Lp_0373) or 20 µl (Lp_2578). Lane M shows the molecular mass standard (kDa); wt indicates supernatant from <i>L. plantarum</i> WCFS1 without expression vector (15 µl).</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Bacterial strains used in this study.

<p>Bacterial strains used in this study.</p

Qualitative functionality of the pSIP inducible gene expression system in <i>Lactobacillus</i> strains grown at various temperatures.

a<p>+, >100 Miller Units (MU) GUS activity; non-induced cultures were <30 MU in all cases.</p>b<p>poor growth at this temperature.</p>c<p>no growth at this temperature.</p

Silver-stained SDS-PAGE gel showing cell-free supernatants of various lactobacilli.

<p>The gel shows NucA production in induced (black arrow) and non-induced cultures (white) of five different <i>Lactobacillus</i> species harboring pLp3050Nuc-SH71. The sample size was 15 µl species except for <i>L. rhamnosus</i> (20 µl). Note that the cultures had different cell densities after the four hour induction period (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091125#pone-0091125-g002" target="_blank">Fig. 2</a>). The horizontal arrow indicates NucA.</p

Plasmid copy number (PCN) in lactobacilli harboring vectors with the <i>NucA</i> reporter gene.

<p>*All plasmid copy numbers were calculated from minimum two biological replicates, each analyzed by triplicate qPCR runs. The data shown are the means ± standard deviations.</p

Silver-stained SDS-PAGE gels showing cell-free supernatants of various lactobacilli.

<p>The gels show NucA production in induced cultures of five different <i>Lactobacillus</i> species harboring (<b>a</b>) pLp3050Nuc-SH71 or (<b>b</b>) pLp0373Nuc-SH71. The sample size was 15 µl except for <i>L. rhamnosus</i> (20 µl). Note that the cultures had different cell densities after the four hour induction period (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091125#pone-0091125-g002" target="_blank">Fig. 2</a>). The lanes marked NucA contain 0.5 µg NucA standard (Sigma). The arrows indicate NucA.</p

Western blots for analysis of secretion efficiency.

<p>The gels show proteins in cell lysates (C) and supernatants (S) of <i>L. plantarum</i> containing pLp3050NucA-SH71 or pLp3050NucA (256_rep replicon), <i>L. curvatus</i>, <i>L. brevis</i> and <i>L. gasseri</i> containing pLp3050NucA-SH71, and <i>L. rhamnosus</i> containing pLp0373NucA-SH71. Lane M, molecular mass standard (20 kDa band); lane NucA contains 0.5 µg NucA standard (Sigma), indicated by the arrow. The lanes marked pEV show supernatants of <i>L. plantarum</i> harboring an empty vector without <i>nucA</i>. For all the culture-derived samples, the sample size corresponded to 20 µl of the original culture, which was harvested 4 hours after induction.</p

Overview of the responses caused by the different experimental factors (ANOVA tables for all biological parameters).

<p>The responses are shown as explained variance (%).</p