scholarly journals Role of the Sortase A in the Release of Cell-Wall Proteinase PrtS in the Growth Medium of Streptococcus thermophilus 4F44

2021 ◽  
Vol 9 (11) ◽  
pp. 2380
Author(s):  
Ahoefa Ablavi Awussi ◽  
Emeline Roux ◽  
Catherine Humeau ◽  
Zeeshan Hafeez ◽  
Bernard Maigret ◽  
...  
Keyword(s):  
Cell Wall ◽  
Lactic Acid ◽  
Lactic Acid Bacterium ◽  
Proteolytic Activity ◽  
Growth Medium ◽  
Cell Envelope ◽  
Streptococcus Thermophilus ◽  
Sortase A ◽  
Cellular Density

Growth of the lactic acid bacterium Streptococcus thermophilus in milk depends on its capacity to hydrolyze proteins of this medium through its surface proteolytic activity. Thus, strains exhibiting the cell envelope proteinase (CEP) PrtS are able to grow in milk at high cellular density. Due to its LPNTG motif, which is possibly the substrate of the sortase A (SrtA), PrtS is anchored to the cell wall in most S. thermophilus strains. Conversely, a soluble extracellular PrtS activity has been reported in the strain 4F44. It corresponds, in fact, to a certain proportion of PrtS that is not anchored to the cell wall but rather is released in the growth medium. The main difference between PrtS of strain 4F44 (PrtS4F44) and other PrtS concerns the absence of a 32-residue imperfect duplication in the prodomain of the CEP, postulated as being required for the maturation and correct subsequent anchoring of PrtS. In fact, both mature (without the prodomain at the N-terminal extremity) and immature (with the prodomain) forms are found in the soluble PrtS4F44 form along with an intact LPNTG at their C-terminal extremity. Investigations we present in this work show that (i) the imperfect duplication is not implied in PrtS maturation; (ii) the maturase PrtM is irrelevant in PrtS maturation which is probably automaturated; and (iii) SrtA allows for the PrtS anchoring in S. thermophilus but the SrtA of strain 4F44 (SrtA4F44) displays an altered activity.

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.

Three distinct proteases are responsible for overall cell surface proteolysis in Streptococcus thermophilus .

2021 ◽  
Author(s):  
Mylène Boulay ◽  
Coralie Metton ◽  
Christine Mézange ◽  
Lydie Oliveira Correia ◽  
Thierry Meylheuc ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Cell Surface ◽  
Lactic Acid Bacterium ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Streptococcus Thermophilus ◽  
Surface Proteins ◽  
Bacterial Vector ◽  
Cell Surface Protease ◽  
Cell Surface Proteolysis

The lactic acid bacterium Streptococcus thermophilus was believed to display only two distinct proteases at the cell surface, namely the cell-envelope protease PrtS and the house-keeping protease HtrA. Using peptidomics, we demonstrate here the existence of an additional active cell-surface protease, which shares significant homology with the SepM protease of Streptococcus mutans . Although all three proteases—PrtS, HtrA, and SepM—are involved in the turnover of surface proteins, they demonstrate distinct substrate specificities. In particular, SepM cleaves proteins involved in cell wall metabolism and cell elongation, and its inactivation has consequences for cell morphology. When all three proteases are inactivated, the residual cell-surface proteolysis of S. thermophilus is approximately 5% of that of the wild-type strain. Importance Streptococcus thermophilus is a lactic acid bacterium widely used as a starter in the dairy industry. Due to its "generally recognized as safe" status and its weak cell-surface proteolytic activity, it is also considered to be a potential bacterial vector for heterologous protein production. Our identification of a new cell surface protease made it possible to construct a mutant strain with a 95% reduction in surface proteolysis, which could be useful in numerous biotechnological applications.

scholarly journals Binding From both sides: TolR and full-length OmpA bind and maintain the local structure of the E. coli cell wall

2018 ◽  
Author(s):  
Alister T. Boags ◽  
Firdaus Samsudin ◽  
Syma Khalid
Keyword(s):  
Cell Wall ◽  
Electrostatic Interactions ◽  
Cell Envelope ◽  
Binding Domain ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
E Coli ◽  
Non Covalent Interactions ◽  
Covalent Interactions

SUMMARYWe present a molecular modeling and simulation study of the of the E. coli cell envelope, with a particular focus on the role of TolR, a native protein of the E. coli inner membrane in interactions with the cell wall. TolR has been proposed to bind to peptidoglycan, but the only structure of this protein thus far is in a conformation in which the putative peptidoglycan binding domain is not accessible. We show that a model of the extended conformation of the protein in which this domain is exposed, binds peptidoglycan largely through electrostatic interactions. We show that non-covalent interactions of TolR and OmpA with the cell wall, from the inner membrane and outer membrane sides respectively, maintain the position of the cell wall even in the absence of Braun’s lipoprotein. When OmpA is truncated to remove the peptidoglycan binding domain, TolR is able to pull the cell wall down towards the inner membrane. The charged residues that mediate the cell-wall interactions of TolR in our simulations, are conserved across a number of species of Gram-negative bacteria.

scholarly journals Global Survey and Genome Exploration of Bacteriophages Infecting the Lactic Acid Bacterium Streptococcus thermophilus

2017 ◽  
Vol 8 ◽  
Author(s):  
Brian McDonnell ◽  
Jennifer Mahony ◽  
Laurens Hanemaaijer ◽  
Horst Neve ◽  
Jean-Paul Noben ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacterium ◽  
Streptococcus Thermophilus ◽  
Global Survey

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.

Role of Enterococci in Cheddar Cheese: Proteolytic Activity and Lactic Acid Development1

1975 ◽  
Vol 38 (1) ◽  
pp. 3-7 ◽  
Author(s):  
JANE P. JENSEN ◽  
G. W. REINBOLD ◽  
C. J. WASHAM ◽  
E. R. VEDAMUTHU
Keyword(s):  
Lactic Acid ◽  
Proteolytic Activity ◽  
Statistical Difference ◽  
Cheddar Cheese ◽  
Protein Breakdown ◽  
Streptococcus Faecalis

Eight lots of Cheddar cheese were manufactured by using two strains of Streptococcus faecalis and Streptococcus durans in combination with a commercial lactic culture. Each lot consisted of a control vat of cheese, manufactured with lactic starter only, and an experimental vat of cheese containing the lactic starter and one of the enterococcus strains. Combinations of two curing temperatures (7.2 and 12.8 C) and two early cooling treatments (air vs. brine cooling) were used for cheeses from each vat to determine the effects of these handling procedures, as well as of enterococcus addition, on proteolysis and lactic acid development. These characteristics were monitored from milling to up to 6 months of curing. Cheeses manufactured with S. faecalis exhibited more protein breakdown than did the control cheeses and those made with S. durans, the latter two being nearly identical in the extent of proteolysis. More proteolysis was consistently observed in those cheeses cured at 12.8 C. No statistical difference was observed inproteolytic activity between air- and brine-cooled cheeses. Cheeses made with S. durans had a higher final percentage of lactic acid than did controls and cheeses made with S. faecalis. Cheeses manufactured with enterococci exhibited a more rapid initial production of lactate. Cheeses cured at 12.8 C had greater percentages of lactic acid compared with those cured at 7.2 C. Air-cooled cheeses also developed significantly higher levels of lactic acid than did brine-cooled cheeses.

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.

scholarly journals Dynamic Changes of Intracellular pH in Individual Lactic Acid Bacterium Cells in Response to a Rapid Drop in Extracellular pH

2000 ◽  
Vol 66 (6) ◽  
pp. 2330-2335 ◽  
Author(s):  
Henrik Siegumfeldt ◽  
K. Björn Rechinger ◽  
Mogens Jakobsen
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Lactic Acid Bacterium ◽  
Intracellular Ph ◽  
Membrane Filter ◽  
Single Cells ◽  
Streptococcus Thermophilus ◽  
Extracellular Ph ◽  
Lactobacillus Delbrueckii ◽  
Rapid Drop

ABSTRACT We describe the dynamics of changes in the intracellular pH (pHi) values of a number of lactic acid bacteria in response to a rapid drop in the extracellular pH (pHex). Strains of Lactobacillus delbrueckii subsp.bulgaricus, Streptococcus thermophilus, andLactococcus lactis were investigated. Listeria innocua, a gram-positive, non-lactic acid bacterium, was included for comparison. The method which we used was based on fluorescence ratio imaging of single cells, and it was therefore possible to describe variations in pHi within a population. The bacteria were immobilized on a membrane filter, placed in a closed perfusion chamber, and analyzed during a rapid decrease in the pHex from 7.0 to 5.0. Under these conditions, the pHi of L. innocua remained neutral (between 7 and 8). In contrast, the pHi values of all of the strains of lactic acid bacteria investigated decreased to approximately 5.5 as the pHex was decreased. No pronounced differences were observed between cells of the same strain harvested from the exponential and stationary phases. Small differences between species were observed with regard to the initial pHi at pHex 7.0, while different kinetics of pHiregulation were observed in different species and also in different strains of S. thermophilus.

scholarly journals Surface Display of the Receptor-Binding Region of the Lactobacillusbrevis S-Layer Protein in Lactococcuslactis Provides Nonadhesive Lactococci with the Ability To Adhere to Intestinal Epithelial Cells

2003 ◽  
Vol 69 (4) ◽  
pp. 2230-2236 ◽  
Author(s):  
Silja Åvall-Jääskeläinen ◽  
Agneta Lindholm ◽  
Airi Palva
Keyword(s):  
Cell Wall ◽  
Lactic Acid ◽  
Epithelial Cells ◽  
Lactic Acid Bacterium ◽  
Receptor Binding ◽  
Surface Display ◽  
Intestinal Epithelial ◽  
Binding Region ◽  
Receptor Binding Region

ABSTRACT Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000. Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer. Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface. In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L. lactis NZ9000. Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene. The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system. These results show that, with the aid of the receptor-binding region of the L. brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium.

Sign in / Sign up

Export Citation Format

Share Document