scholarly journals Characterization of a Novel LysM Domain from Lactobacillus fermentum Bacteriophage Endolysin and Its Use as an Anchor To Display Heterologous Proteins on the Surfaces of Lactic Acid Bacteria

2010 ◽  
Vol 76 (8) ◽  
pp. 2410-2418 ◽  
Author(s):  
Shumin Hu ◽  
Jian Kong ◽  
Wentao Kong ◽  
Tingting Guo ◽  
Mingjie Ji
Keyword(s):  
Cell Wall ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Fluorescent Protein ◽  
Binding Capacity ◽  
Surface Display ◽  
Lactobacillus Fermentum ◽  
Display System ◽  
C Terminus ◽  
Bacteriophage Endolysin

ABSTRACT The endolysin Lyb5, from Lactobacillus fermentum temperate bacteriophage φPYB5, showed a broad lytic spectrum against Gram-positive as well as Gram-negative bacteria. Sequence analysis revealed that the C terminus of the endolysin Lyb5 (Ly5C) contained three putative lysin motif (LysM) repeat regions, implying that Ly5C was involved in bacterial cell wall binding. To investigate the potential of Ly5C for surface display, green fluorescent protein (GFP) was fused to Ly5C at its N or C terminus and the resulting fusion proteins were expressed in Escherichia coli. After being mixed with various cells in vitro, GFP was successfully displayed on the surfaces of Lactococcus lactis, Lactobacillus casei, Lb. brevis, Lb. plantarum, Lb. fermentum, Lb. delbrueckii, Lb. helveticus, and Streptococcus thermophilus cells. Increases in the fluorescence intensities of chemically pretreated L. lactis and Lb. casei cells compared to those of nonpretreated cells suggested that the peptidoglycan was the binding ligand for Ly5C. Moreover, the pH and concentration of sodium chloride were optimized to enhance the binding capacity of GFP-Ly5C, and high-intensity fluorescence of cells was observed under optimal conditions. All results suggested that Ly5C was a novel anchor for constructing a surface display system for lactic acid bacteria (LAB). To demonstrate the applicability of the Ly5C-mediated surface display system, β-galactosidase (β-Gal) from Paenibacillus sp. strain K1, replacing GFP, was functionally displayed on the surfaces of LAB cells via Ly5C. The success in surface display of GFP and β-Gal opened up the feasibility of employing the cell wall anchor of bacteriophage endolysin for surface display in LAB.

The effect of biological additives on the composition and nutritive value of silage

1990 ◽  
Vol 1990 ◽  
pp. 139-139
Author(s):  
M Gonzalez Yanez ◽  
R Mcginn ◽  
D H Anderson ◽  
A R Henderson ◽  
P Phillips
Keyword(s):  
Cell Wall ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Lolium Perenne ◽  
Perennial Ryegrass ◽  
Nutritive Value ◽  
Enzyme System ◽  
Lolium Perenne L ◽  
Grass Silage ◽  
Forage Harvester

It Is claimed that the use of the correct enzyme system as an additive on grass silage will satisfactorily control the fermentation and reduce the cell-wall fibre content, thus preserving the nutrients In the silage and aiding their utilisation by the animal (Henderson and McDonald, 1977; Huhtanen et al, 1985; Raurama et al, 1987; Chamberlain and Robertson, 1989; Gordon, 1989;).The aim of the present experiment was to assess the effect of biological additives, enzymes or a combination of enzymes with an Inoculum of lactic acid bacteria, on the composition of silage and on its nutritive value when offered to store lambs as the sole constituent of their diet.On 1st June 1988, first cut perennial ryegrass (Lolium perenne L) at pre-ear emergence was ensiled direct cut untreated (U), treated with a commercial enzyme (E) or with a commercial inoculum of lactic acid bacteria with enzymes (I) in 6t capacity bunker silos. The grass was cut with a mower and lifted with a New Holland precision chop forage harvester. The additives were pumped onto the grass using a dribble bar sited over the pick-up drum.

scholarly journals A SH3_5 Cell Anchoring Domain for Non-recombinant Surface Display on Lactic Acid Bacteria

2021 ◽  
Vol 8 ◽  
Author(s):  
Pei Kun Richie Tay ◽  
Pei Yu Lim ◽  
Dave Siak-Wei Ow
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Surface Display ◽  
Bioactive Molecules ◽  
Gastric Digestion ◽  
Functional Protein ◽  
Fluorescent Reporter ◽  
Surface Conditions ◽  
Bacterial Surface

Lactic acid bacteria (LAB) are a group of gut commensals increasingly recognized for their potential to deliver bioactive molecules in vivo. The delivery of therapeutic proteins, in particular, can be achieved by anchoring them to the bacterial surface, and various anchoring domains have been described for this application. Here, we investigated a new cell anchoring domain (CAD4a) isolated from a Lactobacillus protein, containing repeats of a SH3_5 motif that binds non-covalently to peptidoglycan in the LAB cell wall. Using a fluorescent reporter, we showed that C-terminal CAD4a bound Lactobacillus fermentum selectively out of a panel of LAB strains, and cell anchoring was uniform across the cell surface. Conditions affecting CAD4a anchoring were studied, including temperature, pH, salt concentration, and bacterial growth phase. Quantitative analysis showed that CAD4a allowed display of 105 molecules of monomeric protein per cell. We demonstrated the surface display of a functional protein with superoxide dismutase (SOD), an antioxidant enzyme potentially useful for treating gut inflammation. SOD displayed on cells could be protected from gastric digestion using a polymer matrix. Taken together, our results show the feasibility of using CAD4a as a novel cell anchor for protein surface display on LAB.

Improving effect of Lactobacillus fermentum TKSN041 on streptozotocin-induced type 2 diabetes in rats

2021 ◽  
Author(s):  
Xianrong Zhou ◽  
Guan-sheng Shang ◽  
Qian Tan ◽  
Qin He ◽  
Xiaoyu Tan ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Lactobacillus Fermentum

With the increasing incidence of type 2 diabetes, it is imperative to identify how to effectively prevent or treat this disease. Studies have shown that some lactic acid bacteria can...

Observation of exopolysaccharides (EPS) from Lactobacillus helveticus SBT2171 using the Tokuyasu method

Microscopy ◽  
2020 ◽  
Vol 69 (5) ◽  
pp. 286-290
Author(s):  
Takamichi Kamigaki ◽  
Akihiro Ogawa
Keyword(s):  
Electron Microscopy ◽  
Cell Wall ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Colloidal Gold ◽  
Ruthenium Red ◽  
Lactobacillus Helveticus ◽  
Ruthenium Red Staining ◽  
Observation Method ◽  
Β Lactoglobulin

Abstract Some species of lactic acid bacteria used for the production of natural cheese produce exopolysaccharides (EPS). Electron microscopy is useful for analyzing the microstructure of EPS produced by lactic acid bacteria. However, pretreatments used to observe the microstructure of EPS by electron microscopy, such as dehydration and resin embedding, can result in EPS flowing out easily from the cell. Therefore, in this study, the Tokuyasu method was conducted on cryosection to reduce EPS outflow. Two types of observation method, namely, using lectin and ruthenium red, were conducted in an attempt to observe EPS produced by Lactobacillus helveticus SBT2171. Observation using the lectin method confirmed that colloidal gold particles conjugated with a lectin recognizing β-galactoside were present in the capsule. Structures that appeared to be β-galactoside-containing slime polysaccharides that were released from the cell wall were also observed. Observation using ruthenium red showed that capsular polysaccharides (CPS) in the capsule were present as a net-like structure. Colloidal gold conjugation with an anti-β-lactoglobulin antibody, in addition to ruthenium red staining, allowed the identification of slime polysaccharides released from the cell wall in the milk protein network derived from the culture medium. Based on these results, the Tokuyasu method was considered to be a useful pretreatment method to clarify and observe the presence of EPS. In particular, both CPS in the capsule and slime exopolysaccharides released from the cell wall were visualized.

scholarly journals Design of a Protein-Targeting System for Lactic Acid Bacteria

2001 ◽  
Vol 183 (14) ◽  
pp. 4157-4166 ◽  
Author(s):  
Y. Dieye ◽  
S. Usai ◽  
F. Clier ◽  
A. Gruss ◽  
J.-C. Piard
Keyword(s):  
Cell Wall ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Protein Targeting ◽  
Medium Optimization ◽  
Cell Envelope ◽  
Staphylococcal Nuclease ◽  
Reporter Protein ◽  
Protein Cell ◽  
Export System

ABSTRACT We designed an expression and export system that enabled the targeting of a reporter protein (the staphylococcal nuclease Nuc) to specific locations in Lactococcus lactis cells, i.e., cytoplasm, cell wall, or medium. Optimization of protein secretion and of protein cell wall anchoring was performed with L.lactis cells by modifying the signals located at the N and C termini, respectively, of the reporter protein. Efficient translocation of precursor (∼95%) is obtained using the signal peptide from the lactococcal Usp45 protein and provided that the mature protein is fused to overall anionic amino acids at its N terminus; those residues prevented interactions of Nuc with the cell envelope. Nuc could be covalently anchored to the peptidoglycan by using the cell wall anchor motif of the Streptococcus pyogenes M6 protein. However, the anchoring step proved to not be totally efficient in L. lactis, as considerable amounts of protein remained membrane associated. Our results may suggest that the defect is due to limiting sortase in the cell. The optimized expression and export vectors also allowed secretion and cell wall anchoring of Nuc in food-fermenting and commensal strains of Lactobacillus. In all strains tested, both secreted and cell wall-anchored Nuc was enzymatically active, suggesting proper enzyme folding in the different locations. These results provide the first report of a targeting system in lactic acid bacteria in which the final location of a protein is controlled and biological activity is maintained.

Antimicrobial Action of Hydrolyzed Chitosan against Spoilage Yeasts and Lactic Acid Bacteria of Fermented Vegetables

2002 ◽  
Vol 65 (5) ◽  
pp. 828-833 ◽  
Author(s):  
TONY SAVARD ◽  
CAROLE BEAULIEU ◽  
ISABELLE BOUCHER ◽  
CLAUDE P. CHAMPAGNE
Keyword(s):  
Molecular Weight ◽  
Cell Wall ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Culture Media ◽  
Antimicrobial Properties ◽  
Spoilage Yeasts ◽  
Entire Cell ◽  
Fermented Vegetables ◽  
Chitosan Lactate

The antimicrobial properties of various chitosan-lactate polymers (ranging from 0.5 to 1.2 MDa in molecular weight) against two yeasts isolated from fermented vegetables and against three lactic acid bacteria from a mixed starter for sauerkraut on methylene blue agar (MBA) and in vegetable juice medium (VJM) were investigated. Chitosan-lactate reduced the growth of all microorganisms in solid (MBA) as well as in liquid (VJM) medium. In MBA, a concentration of 5 g/liter was needed to inhibit the growth of Saccharomyces bayanus, while 1 g/liter was sufficient to inhibit the growth of Saccharomyces unisporus. Lactic acid bacteria were also inhibited in this range of concentrations. The low-molecular-weightchitosan-lactateDP3 (0.5 kDa) was most efficient in solid medium (MBA), and inhibitory activities decreased with increasing hydrolysate lengths. In liquid medium (VJM), 0.5 g of chitosan-lactate per liter reduced the growth rates for both yeasts, but 10 g/liter was insufficient to prevent yeast growth. Intermediate-molecular-weight chitosan-lactate (5 kDa) was more efficient than chitosan of low molecular weight. Native chitosan (1.2 MDa) showed no inhibition in either medium. Microscopic examination of S. unisporus Y-42 after treatment with chitosan-lactate DP25 showed agglutination of a refractive substance on the entire cell wall, suggesting an interaction between chitosan and the cell wall. When chitosanase was added to the culture media containing chitosan-lactate, refractive substances could not be observed.

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