Heterologous Expression of Mannanase and Developing a New Reporter Gene System in <i>Lactobacillus casei</i> and <i>Escherichia coli</i>

<div><p>Reporter gene systems are useful for studying bacterial molecular biology, including the regulation of gene expression and the histochemical analysis of protein products. Here, two genes, β-1,4-mannanase (<i>manB</i>) from <i>Bacillus pumilus</i> and β-glucuronidase (<i>gusA</i>) from <i>Escherichia coli</i> K12, were cloned into the expression vector pELX1. The expression patterns of these reporter genes in <i>Lactobacillus casei</i> were investigated by measuring their enzymatic activities and estimating their recombinant protein yields using western blot analysis. Whereas mannanase activity was positively correlated with the accumulation of ManB during growth, GusA activity was not; western blot analysis indicated that while the amount of GusA protein increased during later growth stages, GusA activity gradually decreased, indicating that the enzyme was inactive during cell growth. A similar trend was observed in <i>E</i>. <i>coli</i> JM109. We chose to use the more stable mannanase gene as the reporter to test secretion expression in <i>L</i>. <i>casei</i>. Two pELX1-based secretion vectors were constructed: one carried the signal peptide of the unknown secretion protein Usp45 from <i>Lactococcus lactis</i> (pELSH), and the other contained the full-length SlpA protein from the S-layer of <i>L</i>. <i>acidophilus</i> (pELWH). The secretion of ManB was detected in the supernatant of the pELSH-ManB transformants and in the S-layer of the cell surface of the pELWH-ManB transformants. This is the first report demonstrating that the <i>B</i>. <i>pumilus manB</i> gene is a useful reporter gene in <i>L</i>. <i>casei</i> and <i>E</i>.<i>coli</i>.</p></div

SDS-PAGE and western blot of His-tagged recombinant proteins in <i>L</i>. <i>casei</i> MCJΔ1.

<p>(<b>A</b>) SDS-PAGE analysis. Lane M, marker. Lanes 1–3, whole protein extracts of the pELX1, pELX1-ManB and pELX1-GusA transformants. (<b>B</b>) Western blot analysis, which suggested that both recombinant proteins were expressed. Lane M, marker. Lanes 1–3, whole protein extracts of the pELX1, pELX1-ManB and pELX1-GusA transformants. The protein concentrations of all samples were normalized to 3 mg·ml^-1, and the sample volume loaded was15 μl in both the SDS-PAGE and western blot analysis.</p

Bacterial diversity index calculated from the DGGE banding patterns (Fig. 1A).

<p>N (negative control, basal diet); P (positive control, diet supplemented with neomycin); L, M, H (diets supplemented with probiotics 0.5×10⁹, 1.0×10⁹ and 2.5×10⁹ CFU/kg feed, respectively);</p><p>*1/D, the reciprocal of Simpson diversity index.</p><p>Bacterial diversity index calculated from the DGGE banding patterns (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116635#pone.0116635.g001" target="_blank">Fig. 1A</a>).</p

Western blot detection of ManB and GusA in <i>L</i>. <i>casei</i>.

<p>The final concentration of all the WCP samples were adjusted to 0.2 mg·ml^-1 for ManB samples and 0.1 mg·ml^-1 for GusA samples, and 10-μl aliquots were added in each lane of the electrophoresis gels. (<b>A</b>) Western blot of His-tagged ManB from the <i>L</i>. <i>casei</i> MCJΔ1 transformant. These results show a similar increase in the amount of ManB protein and ManB activity during early exponential growth (until OD₆₀₀ = 1.8). Lane M, marker. Lanes 1–6, ManB from the <i>L</i>. <i>casei</i> MCJΔ1 transformant at different cell densities (OD₆₀₀ = 1.0, 1.8, 2.9, 4.3, 5.0 and 6.7). (<b>B</b>) Western blot of His-tagged GusA from the <i>L</i>. <i>casei</i> MCJΔ1 transformant. These results show that although GusA activity decreased rapidly from the exponential growth phase to the stationary growth phase, the western blot analysis of GusA revealed large amounts of this protein in late growth-phase cell samples, when the specific activity of the enzyme dropped markedly. Lane M, marker. Lanes 1–6, GusA of the <i>L</i>. <i>casei</i> MCJΔ1 transformant at different cell densities (OD₆₀₀ = 1.0, 2.2, 3.6, 4.6, 5.8 and 6.6).</p

<i>Lactobacillus</i> community of weaned piglets fed with neomycin or <i>E. faecalis</i>.

<p>(A) DGGE profiles of V3 region of the 16S rDNA gene fragments with the primes Lac1 and Lac2-GC. The denaturant gradient range is from 41% to 60%. Lanes N (negative control, basal diet); P (positive control, diet supplemented with neomycin); L, M, H (diets supplemented with probiotics 0.5×10⁹, 1.0×10⁹ and 2.5×10⁹ CFU/kg feed, respectively); (B) UPGMA cluster analysis of Dice similarity indices from DGGE profiles.</p

Expression and secretion of the ManB-SlpA fusion protein in <i>L</i>. <i>casei</i> MCJΔ1/ pELWH-ManB.

<p><b>(A)</b> S-layer-bound mannanase activity from the functional fusion protein on the cell surface of <i>L</i>. <i>casei</i> MCJΔ1/pELWH-ManB. The mannanase activity increased during the early growth phase, peaking at OD₆₀₀ = 3.7 with an activity of 28.4 mU·5×10⁹ cfu^-1, and then decreased. <b>(B)</b> SDS-PAGE and western blot analysis of the composition of the S-layer proteins. <i>L</i>. <i>casei</i> MCJΔ1 and <i>L</i>. <i>casei</i> MCJΔ1/pELWH were used as controls (the OD₆₀₀ of all cell samples was 3.0). The fusion protein was barely detectable in <i>L</i>. <i>casei</i> MCJΔ1/pELWH-ManB (Lane 6), as reflected by a very low mannanase activity on the cell surface. Lane M, Marker. Lanes 1–3, SDS-PAGE-analyzed S-layer proteins from <i>L</i>. <i>casei</i> MCJΔ1, <i>L</i>. <i>casei</i> MCJΔ1/pELWH and <i>L</i>. <i>casei</i> MCJΔ1/pELWH-ManB. Lanes 4–6, western blot of His-tagged target proteins from the S-layer proteins of <i>L</i>. <i>casei</i> MCJΔ1, <i>L</i>. <i>casei</i> MCJΔ1/pELWH and <i>L</i>. <i>casei</i> MCJΔ1/pELWH-ManB. The red arrow represents the target proteins. Error bars represent standard errors from three replicate experiments.</p

Expression and secretion of ManB in <i>L</i>. <i>casei</i> MCJΔ1/pELSH-ManB.

<p>(<b>A</b>) ManB activity in the supernatant. ManB activity reached 8.85 U·ml^-1 during the exponential growth phase of <i>L</i>. <i>casei</i> MCJΔ1/pELSH-ManB and remained relatively constant above 8 U·ml^-1 throughout the late growth phase (between OD₆₀₀ = 1.9 to 4.8) before decreasing. (<b>B</b>) Western blot of His-tagged ManB in the supernatant. Aliquots of 10 μl were added in each lane of the electrophoresis gels. These results clearly showed that secreted mannanase activity was correlated with the accumulation of ManB during cell growth. Lane M, marker. Lanes 1–8, ManB of the <i>L</i>. <i>casei</i> MCJΔ1 transformant at different cell densities (OD₆₀₀ = 0.5, 0.9, 1.9, 2.8, 3.4, 4.8, 5.8 and 6.2). Error bars represent standard errors from three replicate experiments.</p

Dietary <i>Enterococcus faecalis</i> LAB31 Improves Growth Performance, Reduces Diarrhea, and Increases Fecal <i>Lactobacillus</i> Number of Weaned Piglets

<div><p>Lactic acid bacteria (LAB) have been shown to enhance performance of weaned piglets. However, few studies have reported the addition of LAB <i>Enterococcus faecalis</i> as alternatives to growth promoting antibiotics for weaned piglets. This study evaluated the effects of dietary <i>E. faecalis</i> LAB31 on the growth performance, diarrhea incidence, blood parameters, fecal bacterial and <i>Lactobacillus</i> communities in weaned piglets. A total of 360 piglets weaned at 26 ± 2 days of age were randomly allotted to 5 groups (20 pens, with 4 pens for each group) for a trial of 28 days: group N (negative control, without antibiotics or probiotics); group P (Neomycin sulfate, 100 mg/kg feed); groups L, M and H (supplemented with <i>E. faecalis</i> LAB31 0.5×10⁹, 1.0×10⁹, and 2.5×10⁹ CFU/kg feed, respectively). Average daily gain and feed conversion efficiency were found to be higher in group H than in group N, and showed significant differences between group H and group P (<i>P₀</i> < 0.05). Furthermore, groups H and P had a lower diarrhea index than the other three groups (<i>P₀</i> < 0.05). Denaturing gradient gel electrophoresis (DGGE) showed that the application of probiotics to the diet changed the bacterial community, with a higher bacterial diversity in group M than in the other four groups. Real-time PCR revealed that the relative number of <i>Lactobacillus</i> increased by addition of probiotics, and was higher in group H than in group N (<i>P₀</i> < 0.05). However, group-specific PCR-DGGE showed no obvious difference among the five groups in <i>Lactobacillus</i> composition and diversity. Therefore, the dietary addition of <i>E. faecalis</i> LAB31 can improve growth performance, reduce diarrhea, and increase the relative number of <i>Lactobacillus</i> in feces of weaned piglets.</p></div

Identification of band fragments in DGGE gels (Fig. 1A).

<p>* Bands are numbered according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116635#pone.0116635.g001" target="_blank">Fig. 1A</a>.</p><p>^◆Identity represents the sequence identity (%) compared with that in the GenBank database.</p><p>Identification of band fragments in DGGE gels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116635#pone.0116635.g001" target="_blank">Fig. 1A</a>).</p

Expression of ManB and GusA in <i>L</i>. <i>casei</i> MCJΔ1.

<p>In the growth curves, <i>L</i>. <i>casei</i> MCJΔ1 showed exponential growth until OD₆₀₀ = 1.8, and the stationary phase was from OD₆₀₀ = 1.8 to 6.8. In the growth curve, there were no significant difference between the negative control <i>L</i>. <i>casei</i> MCJΔ1 pELX1 transformant, the <i>L</i>. <i>casei</i> MCJΔ1 ManB transformant and the <i>L</i>. <i>casei</i> MCJΔ1 GusA transformant. the OD₆₀₀ in the activity curve of <i>L</i>. <i>casei</i> MCJΔ1/pELX1-ManB transformant were 0.2, 0.5, 1.0, 1.8, 2.9, 4.3, 5.0 and 6.7, and those of <i>L</i>. <i>casei</i> MCJΔ1/pELX1-GusA transformant were 0.2, 0.5, 1.0, 2.2, 3.6, 4.6, 5.8 and 6.6, respectively. The specific activity of ManB increased in the exponential growth phase until the OD₆₀₀ of the culture reached 1.8 (23 U·mg^-1 WCP). Then, ManB activity decreased gradually to 15 U·mg^-1 WCP in the late growth phase before remaining relatively constant. The specific activity of GusA peaked during the exponential growth phase (OD₆₀₀ = 1.0, 75 U·mg^-1 WCP) before decreasing rapidly in the late growth phase by more than 60% (OD₆₀₀ = 1.0 versus 4.6). Error bars represent standard errors from three replicate experiments.</p