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.

scholarly journals Streptococcus thermophilus Cell Wall-Anchored Proteinase: Release, Purification, and Biochemical and Genetic Characterization

2000 ◽  
Vol 66 (11) ◽  
pp. 4772-4778 ◽  
Author(s):  
María Dolores Fernandez-Espla ◽  
Peggy Garault ◽  
Véronique Monnet ◽  
Françoise Rul
Keyword(s):  
Cell Wall ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Cell Envelope ◽  
Serine Proteinase ◽  
Streptococcus Thermophilus ◽  
Alumina Powder ◽  
A Cell ◽  
Cell Envelopes

ABSTRACT Streptococcus thermophilus CNRZ 385 expresses a cell envelope proteinase (PrtS), which is characterized in the present work, both at the biochemical and genetic levels. Since PrtS is resistant to most classical methods of extraction from the cell envelopes, we developed a three-step process based on loosening of the cell wall by cultivation of the cells in the presence of glycine (20 mM), mechanical disruption (with alumina powder), and enzymatic treatment (lysozyme). The pure enzyme is a serine proteinase highly activated by Ca2+ ions. Its activity was optimal at 37°C and pH 7.5 with acetyl-Ala-Ala-Pro-Phe-paranitroanilide as substrate. The study of the hydrolysis of the chromogenic and casein substrates indicated that PrtS presented an intermediate specificity between the most divergent types of cell envelope proteinases from lactococci, known as the PI and PIII types. This result was confirmed by the sequence determination of the regions involved in substrate specificity, which were a mix between those of PI and PIII types, and also had unique residues. Sequence analysis of the PrtS encoding gene revealed that PrtS is a member of the subtilase family. It is a multidomain protein which is maturated and tightly anchored to the cell wall via a mechanism involving an LPXTG motif. PrtS bears similarities to cell envelope proteinases from pyogenic streptococci (C5a peptidase and cell surface proteinase) and lactic acid bacteria (PrtP, PrtH, and PrtB). The highest homologies were found with streptococcal proteinases which lack, as PrtS, one domain (the B domain) present in cell envelope proteinases from all other lactic acid bacteria.

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.

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.

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.

Antibiotic susceptibility of different lactic acid bacteria strains

2011 ◽  
Vol 2 (4) ◽  
pp. 335-339 ◽  
Author(s):  
N. Karapetkov ◽  
R. Georgieva ◽  
N. Rumyan ◽  
E. Karaivanova
Keyword(s):  
Cell Wall ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Lactobacillus Acidophilus ◽  
Streptococcus Thermophilus ◽  
Lactobacillus Helveticus ◽  
Lactobacillus Delbrueckii ◽  
Cell Wall Synthesis ◽  
High Susceptibility ◽  
Lactobacillus Delbrueckii Subsp

Five lactic acid bacteria (LAB) strains belonging to species Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis and Streptococcus thermophilus were tested for their susceptibility to 27 antibiotics. The minimum inhibitory concentrations of each antimicrobial were determined using a microdilution test. Among the strains a high susceptibility was detected for most of the cell-wall synthesis inhibitors (penicillins, cefoxitin and vancomycin) and resistance toward inhibitors of DNA synthesis (trimethoprim/sulfonamides and fluoroquinolones). Generally, the Lactobacillus strains were inhibited by antibiotics such as chloramphenicol, erythromycin and tetracycline at breakpoint levels lower or equal to the levels defined by the European Food Safety Authority. Despite the very similar profile of S. thermophilus LC201 to lactobacilli, the detection of resistance toward erythromycin necessitates the performance of additional tests in order to prove the absence of transferable resistance genes.

Proinflammatory Cytokine and Nitric Oxide Induction in Murine Macrophages by Cell Wall and Cytoplasmic Extracts of Lactic Acid Bacteria

1999 ◽  
Vol 62 (12) ◽  
pp. 1435-1444 ◽  
Author(s):  
MARIA VICTORIA TEJADA-SIMON ◽  
JAMES J. PESTKA
Keyword(s):  
Nitric Oxide ◽  
Cell Wall ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Cell Walls ◽  
Streptococcus Thermophilus ◽  
No Production ◽  
Peyer’S Patch ◽  
Peyer's Patch

Cells from a number of bacterial genera have been shown to possess mitogenic and polyclonal activating properties when cultured with cells of the immune system. Based on previously reported health immune-enhancing effects of fermented dairy products, we tested the potentiating effects of representative lactic acid bacteria and their extracts on leukocyte function. Specifically, the effects of in vitro exposure to heat-killed cells of Bifidobacterium, Lactobacillus acidophilus, L. bulgaricus, L. casei, L. gasseri, L. helveticus, L. reuteri, and Streptococcus thermophilus, their cell walls, and their cytoplasmic extracts on proliferation as well as cytokine and nitric oxide (NO) production were examined in the RAW 264.7 macrophage cell line. A similar strategy was applied to murine cultures composed of peritoneal, spleen, and Peyer's patch cells. Both the cell wall and cytoplasmic fractions of lactic acid bacteria were able to stimulate cloned macrophages to produce significant amounts of tumor necrosis factor-α, (interleukin) IL-6, and NO. Pronounced enhancement of IL-6 production by peritoneal cells was observed when cultured with those extracts, whereas, effects were not noted in spleen and Peyer's patch cell cultures from mice. Based on the results, it appears that, as a group, the lactic acid bacteria were capable of stimulating macrophages and possibly other immune cells to produce cytokines and NO, and both their cell walls and cytoplasm contributed to these capacities.

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