1,137 research outputs found

    Obtaining and Characteristic of the Autolysate of Lactic Acid Bacteria

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    Fragments of bacterial peptidoglycan relating to compounds of the muropeptide series have high biological activity. They have an immunological effect, due to the fact that they are signals for immune receptors and, accordingly, their activators. In order to obtain fragments of peptidoglycans, the autolysis of the bacterial mass Lactobacillus delbrueckii subsp. Bulgaricus 2–11 was performed and the characteristics of protein nature products of autolysate were given. To obtain cell suspensions at different growth stages, a growth curve of the bacterial culture was constructed for 48 hours. The autolysis of biomass was carried out at the end of the logarithmic phase of growth at the temperatures 37–90 0C and after the stationary phase of growth at the temperatures 37–90 0C for 7 days. The degree of autolytic changes was evaluated by the definition of colony forming units, the accumulation of amino acids, low molecular weight peptides and soluble protein in the autolysate. The results of studies of autolytic biomass changes have shown that the largest accumulation of amino acids occurs in autolysate, which was obtained at the end of the logarithmic phase of the growth of bacterial mass at exposures at 90 0C. The molecular-mass composition of protein nature compounds of the autolysate soluble fraction is determined. It is established that peptides with a molecular weight in the range corresponding to the molecular weight of the muropeptides are presented in the autolystate, but their quantity is rather insignificant

    Evaluation of a microbial muramidase supplementation on growth performance, apparent ileal digestibility, and intestinal histology of broiler chickens

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    The current study evaluated the effects of different inclusion levels of microbial muramidase (Muramidase 007, DSM Nutritional Products) on gastrointestinal functionality by determination of apparent ileal digestibility (AID) of nutrients, investigation of intestinal histomorphology, and quantification of resulting growth performance. Four maize-wheat-soybean experimental diets were produced without (C) and with different dosages of muramidase: low (L, 25,000 LSU(F)/kg), medium (M, 35,000 LSU(F)/kg), and high (H, 45,000 LSU(F)/kg); diets were fed to broilers for 35 d. At the end of the experiment, AID of ether extract (EE), crude protein (CP), Ca, and P were determined and samples of the mid-jejunum and -ileum were collected for histomorphological observations. Data were subjected to ANOVA analysis using the GLM procedure. Orthogonal polynomial contrasts were used to assess linear and quadratic effects of different levels of the muramidase. At the end of the trial, Muramidase 007 supplementation linearly increased body weight gain and decreased feed conversion ratio (FCR) (P ≤ 0.05). Adding the muramidase to broiler diets also linearly increased the European poultry efficiency factor (P ≤ 0.05). Inclusion of the muramidase in broiler diets linearly increased AID of CP, EE, and P (P ≤ 0.05), and the H group had a higher AID of EE and CP compared to C group (P ≤ 0.05). Microbial muramidase supplementation linearly increased ileal villus length to crypt depth ratio and decreased the number of ileal CD45 cells (P ≤ 0.05). Broilers fed M and H diets had fewer number of CD45 cells in the ileum compared to those in C group (P ≤ 0.05). In conclusion, the results of the present study demonstrated that inclusion of the microbial muramidase in broiler diets could increase AID of key nutrients and improve growth performance in broilers. Adding microbial muramidase to broiler diets can therefore be considered as an interesting prospect to improve gastrointestinal functionality. Biological mechanisms causing these improvements need to be studied further

    Regulation and Molecular Basis of Environmental Muropeptide Uptake and Utilization in Fastidious Oral Anaerobe Tannerella forsythia

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    Tannerella forsythia is a Gram-negative oral anaerobe associated with periodontitis. This bacterium is auxotrophic for the peptidoglycan amino sugar N-acetylmuramic (MurNAc) and likely relies on scavenging peptidoglycan fragments (muropeptides) released by cohabiting bacteria during their cell wall recycling. Many Gram-negative bacteria utilize an inner membrane permease, AmpG, to transport peptidoglycan fragments into their cytoplasm. In the T. forsythia genome, the Tanf_08365 ORF has been identified as a homolog of AmpG permease. In order to confirm the functionality of Tanf_08365, a reporter system in an Escherichia coli host was generated that could detect AmpG-dependent accumulation of cytosolic muropeptides via a muropeptide-inducible β-lactamase reporter gene. In trans complementation of this reporter strain with a Tanf_08365 containing plasmid caused significant induction of β-lactamase activity compared to that with an empty plasmid control. These data indicated that Tanf_08365 acted as a functional muropeptide permease causing accumulation of muropeptides in E. coli and thus suggested that it is a permease involved in muropeptide scavenging in T. forsythia. Furthermore, we showed that the promoter regulating the expression of Tanf_08365 was activated significantly by a hybrid two-component system regulatory protein, GppX. We also showed that compared to the parental T. forsythia strain a mutant lacking GppX in which the expression of AmpG was reduced significantly attenuated in utilizing free muropeptides. In summary, we have uncovered the mechanism by which this nutritionally fastidious microbe accesses released muropeptides in its environment, opening up the possibility of targeting this activity to reduce its numbers in periodontitis patients with potential benefits in the treatment of disease

    Mutations in the Lipopolysaccharide Biosynthesis Pathway Interfere with Crescentin-Mediated Cell Curvature in Caulobacter crescentus

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    Bacterial cell morphogenesis requires coordination among multiple cellular systems, including the bacterial cytoskeleton and the cell wall. In the vibrioid bacterium Caulobacter crescentus, the intermediate filament-like protein crescentin forms a cell envelope-associated cytoskeletal structure that controls cell wall growth to generate cell curvature. We undertook a genetic screen to find other cellular components important for cell curvature. Here we report that deletion of a gene (wbqL) involved in the lipopolysaccharide (LPS) biosynthesis pathway abolishes cell curvature. Loss of WbqL function leads to the accumulation of an aberrant Opolysaccharide species and to the release of the S layer in the culture medium. Epistasis and microscopy experiments show that neither S-layer nor O-polysaccharide production is required for curved cell morphology per se but that production of the altered O-polysaccharide species abolishes cell curvature by apparently interfering with the ability of the crescentin structure to associate with the cell envelope. Our data suggest that perturbations in a cellular pathway that is itself fully dispensable for cell curvature can cause a disruption of cell morphogenesis, highlighting the delicate harmony among unrelated cellular systems. Using the wbqL mutant, we also show that the normal assembly and growth properties of the crescentin structure are independent of its association with the cell envelope. However, this envelope association is important for facilitating the local disruption of the stable crescentin structure at the division site during cytokinesis

    Peptidoglycan editing provides immunity to Acinetobacter baumannii during bacterial warfare

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    Peptidoglycan (PG) is essential in most bacteria. Thus, it is often targeted by various assaults, including interbacterial attacks via the type VI secretion system (T6SS). Here, we report that the Gram-negative bacteriu

    Antibiotic Resistance and Cell-Wall Recycling in Pseudomonas aeruginosa

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    The threat of antibiotic resistance and the global rise of pan-resistant bacteria is a serious concern at present. Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen is frequently associated with multi and pan-drug resistant infections. This research delves into the mechanism of resistance to a class of drugs known as the β-lactams. AmpC β-lactamase encoded chromosomally in P. aeruginosa is one of the predominant causes of resistance to many β-lactams. Previous research on this pathway identified the AmpC regulatory protein - AmpR and elaborated on its regulon in P. aeruginosa. In this dissertation, further investigation in the mechanisms associated with AmpR regulation of AmpC and its connection with the cell-wall recycling pathway is explored. Cell-wall recycling, a common phenomenon in both Gram-positive and negative bacteria is investigated in some detail in P. aeruginosa for the first time. The identity of the cell-wall recycling products or muropeptides in P. aeruginosa is elucidated. Around 20 distinct muropeptides were identified through liquid chromatography/mass spectrometry analyses of bacterial extracts. Furthermore, iv the muropeptide effector of AmpR that is instrumental in the activation of this transcription factor is identified. The role of two permeases AmpG and AmpP in antibiotic resistance and cell-wall recycling are also investigated by comparing antibiotic susceptibility and muropeptide profile of the isogenic mutants of ampG and ampP with the wild-type PAO1. Along with investigating permeases, the role of a putative N-acetylglucosaminidase FlgJ is also investigated. Finally, keeping in mind the broad role of AmpR in regulating P. aeruginosa virulence and antibiotic resistance, we try to identify small -molecule inhibitors for AmpR. In our effort to identify inhibitors, a novel reporter-based screening assay is developed. In summary, this dissertation increases our understanding of cell-wall recycling and mechanisms of β-lactam resistance and attempts at establishing novel-antibacterial targets and inhibitors

    AcmA of Lactococcus lactis is an N-acetylglucosaminidase with an optimal number of LysM domains for proper functioning

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    AcmA, the major autolysin of Lactococcus lactis MG1363 is a modular protein consisting of an N-terminal active site domain and a C-terminal peptidoglycan-binding domain. The active site domain is homologous to that of muramidase-2 of Enterococcus hirae, however, RP-HPLC analysis of muropeptides released from Bacillus subtilis peptidoglycan, after digestion with AcmA, shows that AcmA is an N-acetylglucosaminidase. In the C-terminus of AcmA three highly similar repeated regions of 45 amino acid residues are present, which are separated by short nonhomologous sequences. The repeats of AcmA, which belong to the lysine motif (LysM) domain family, were consecutively deleted, removed, or, alternatively, one additional repeat was added, without destroying the cell wall-hydrolyzing activity of the enzyme in vitro, although AcmA activity was reduced in all cases. In vivo, proteins containing no or only one repeat did not give rise to autolysis of lactococcal cells, whereas separation of the producer cells from the chains was incomplete. Exogenously added AcmA deletion derivatives carrying two repeats or four repeats bound to lactococcal cells, whereas the derivative with no or one repeat did not. In conclusion, these results show that AcmA needs three LysM domains for optimal peptidoglycan binding and biological functioning
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