scholarly journals Cloning and expression of the bacteriophage-derived endolysin against Aeromonas hydrophila

2021 ◽  
Vol 947 (1) ◽  
pp. 012035
Author(s):  
Nguyen Tan Loc ◽  
Bui Thanh Huyen ◽  
Hoang Anh Hoang ◽  
Le Phi Nga
Keyword(s):  
Cell Wall ◽  
Aeromonas Hydrophila ◽  
Antibacterial Agent ◽  
Cloning And Expression ◽  
Lytic Phage ◽  
Bacterial Cells ◽  
E Coli ◽  
Cell Wall Hydrolase ◽  
Peptidoglycan Hydrolases ◽  
Striped Catfish

Abstract Hemorrhagic septicemia disease in striped catfish is caused by Aeromonas hydrophila bacterium. Antibiotics are commonly used to treat this disease, however, due to antibiotic resistance in A. hydrophila, it is necessary to have an alternative antibacterial agent to antibiotics. Endolysins are bacteriophage-encoded peptidoglycan hydrolases that are synthesized at the end of the lytic phage replication cycle, they lyse the host bacterial cell wall and release new bacteriophage virions. In this study, an endolysin (cell wall hydrolase) derived from A. hydrophila phage PVN02 was artificially synthesized, cloned into pET28a(+) and successfully expressed in E. coli BL21 (DE3). The recombinant endolysin, cell wall hydrolase strongly exhibited antimicrobial activity against A. hydrophila with a reduction of 3-log CFU/ml of A. hydrophila after 30 minutes of mixing and further 30 minutes of incubation, the bacterial cells were lysed completely. It should be emphasized that the lytic activity by the recombinant endolysin to A. hydrophila bacteria did not require a pretreatment with an outer-membrane permeabilizer. The results of our study showed a potential of use this recombinant endolysin as a novel antibacterial agent to replace antibiotics in the treatment of hemorrhagic septicemia diseases in striped catfish.

Analysis of the Crushing Effect about Ultrasound on E. coli and B. subtilis

2012 ◽  
Vol 260-261 ◽  
pp. 1017-1021
Author(s):  
Xin Ying Wang ◽  
Yong Tao Liu ◽  
Min Hui ◽  
Ji Fei Xu
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Bacillus Subtilis ◽  
Bacterial Cell ◽  
Logarithmic Phase ◽  
Growth Stages ◽  
Bacterial Cells ◽  
E Coli ◽  
Different Growth Stages ◽  
Main Factor

Escherichia coli and Bacillus subtilis as objects of the study, ultrasonic fragmentation acted on the bacterial cells in different growth stages, results showed that, it’s similar to the crushing effect of ultrasound on E. coli and B. subtilis cells of different growth stages, the highest crushing rate in the logarithmic phase, reached to 95.8% and 94.3% respectively, the crushing rate of adjustment phase is lowest, maintained at around 60%, the crushing rate stability cell was centered, which can be achieved 90%. The structure of the bacterial cell wall didn’t the main factor to decide the ultrasonic fragmentation effect, but different growth periods of bacterial cells did the determinant.

scholarly journals Phage Mediate Bacterial Self Recognition

2018 ◽  
Author(s):  
Sooyeon Song ◽  
Yunxue Guo ◽  
Jun-Seob Kim ◽  
Xiaoxue Wang ◽  
Thomas K. Wood
Keyword(s):  
Kin Recognition ◽  
Group Behavior ◽  
Lytic Phage ◽  
Bacterial Cells ◽  
Demarcation Line ◽  
E Coli ◽  
Self Recognition ◽  
The Family ◽  
K 12

AbstractCells are social, and self-recognition is an important and conserved aspect of group behavior where cells assist kin and antagonize non-kin to conduct group behavior such as foraging for food and biofilm formation. However, the role of the common bacterial cohabitant, phage, in kin recognition, has not been explored. Here we find that a boundary (demarcation line) is formed between different swimmingEscherichia colistrains but not between identical clones; hence, motile bacterial cells discriminate between self and non-self. The basis for this self-recognition is a novel, 49 kb, T1-type, lytic phage of the family siphoviridae (named here SW1) that controls formation of the demarcation line by utilizing one of the host’s cryptic prophage proteins, YfdM, to propagate. Critically, SW1 increases the fitness ofE. coliK-12 compared to the identical strain that lacks the phage. Therefore, bacteria use phage to recognize kin.

POLYETHYLENE GLYCOL-FUNCTIONALIZED MAGNETIC (Fe3O4) NANOPARTICLES: A GOOD METHOD FOR A SUCCESSFUL ANTIBACTERIAL THERAPEUTIC AGENT VIA DAMAGE DNA MOLECULE

2019 ◽  
Vol 26 (10) ◽  
pp. 1950079 ◽  
Author(s):  
WALEED K. ABDUL KADHIM ◽  
UDAY M. NAYEF ◽  
MAJID S. JABIR
Keyword(s):  
Cell Wall ◽  
Antibacterial Activity ◽  
Polyethylene Glycol ◽  
Nucleic Acid ◽  
Antibacterial Agent ◽  
Bacterial Species ◽  
Good Method ◽  
Diffusion Method ◽  
Bacterial Cells ◽  
Average Size

Magnetite (Fe3O4) nanoparticles (MPs) capped with polyethylene glycol (PEG) were prepared by a hydrothermal method, and their antibacterial activity was examined against Staphylococcus aureus, Escherichia coli and Psudomonas aeruginosa. The functionalized NPs were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), vibrating sample magnetometer (VSM), Fourier transform infrared (FTIR) spectroscopy, and Thermogravimetry (TG). The average size of the Fe3O4 was in the range 9–20[Formula: see text]nm, while the functionalized PEG–Fe3O4 had an average size of 5–15[Formula: see text]nm. The PEG–Fe3O4 exhibited superparamagnetism and high saturation magnetization at room temperature. The antibacterial activity of the Fe3O4 and PEG–Fe3O4 were evaluated against E. coli, S. aureus, and P. aeruginosa using the agar well diffusion method. The changes in the morphology of the studied bacterial species were observed via SEM, while the mode of action of the studied agents was determined via the detection of reactive oxygen species (ROS) using Acridine orange-ethidium bromide (AO/EtBr) staining method. The results showed that PEG-functionalized magnetic (Fe3O4) NPs as a novel DNA-mediated antibacterial agent. The PEG–Fe3O4 NPs were observed to destroy the bacterial cells by permeating the bacterial nucleic acid and cytoplasmic membrane, resulting in the loss of cell-wall integrity, nucleic acid damage, and increased cell-wall permeability. The PEG–Fe3O4 NPs could serve as a potential antibacterial agent in future biomedical and pharmaceutical applications.

scholarly journals Conserved mechanism of cell-wall synthase regulation revealed by the identification of a new PBP activator inPseudomonas aeruginosa

2018 ◽  
Vol 115 (12) ◽  
pp. 3150-3155 ◽  
Author(s):  
Neil G. Greene ◽  
Coralie Fumeaux ◽  
Thomas G. Bernhardt
Keyword(s):  
Cell Wall ◽  
Drug Targets ◽  
Bacterial Cells ◽  
Acinetobacter Baylyi ◽  
Gram Negative ◽  
E Coli ◽  
Outer Membrane Lipoprotein ◽  
Synthase Regulation

Penicillin-binding proteins (PBPs) are synthases required to build the essential peptidoglycan (PG) cell wall surrounding most bacterial cells. The mechanisms regulating the activity of these enzymes to control PG synthesis remain surprisingly poorly defined given their status as key antibiotic targets. Several years ago, the outer-membrane lipoproteinEcLpoB was identified as a critical activator ofEscherichia coliPBP1b (EcPBP1b), one of the major PG synthases of this organism. Activation ofEcPBP1b is mediated through the association ofEcLpoB with a regulatory domain onEcPBP1b called UB2H. Notably,Pseudomonas aeruginosaalso encodes PBP1b (PaPBP1b), which possesses a UB2H domain, but this bacterium lacks an identifiable LpoB homolog. We therefore searched for potentialPaPBP1b activators and identified a lipoprotein unrelated to LpoB that is required for the in vivo activity ofPaPBP1b. We named this protein LpoP and found that it interacts directly withPaPBP1b in vitro and is conserved in many Gram-negative species. Importantly, we also demonstrated thatPaLpoP-PaPBP1b as well as an equivalent protein pair fromAcinetobacter baylyican fully substitute forEcLpoB-EcPBP1b inE. colifor PG synthesis. Furthermore, we show that amino acid changes inPaPBP1b that bypass thePaLpoP requirement map to similar locations in the protein as changes promotingEcLpoB bypass inEcPBP1b. Overall, our results indicate that, although different Gram-negative bacteria activate their PBP1b synthases with distinct lipoproteins, they stimulate the activity of these important drug targets using a conserved mechanism.

scholarly journals A MurF Inhibitor That Disrupts Cell Wall Biosynthesis in Escherichia coli

2007 ◽  
Vol 51 (12) ◽  
pp. 4420-4426 ◽  
Author(s):  
Ellen Z. Baum ◽  
Steven M. Crespo-Carbone ◽  
Alexandra Klinger ◽  
Barbara D. Foleno ◽  
Ignatius Turchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Antibacterial Activity ◽  
Binding Assay ◽  
Pharmacophore Model ◽  
Fold Increase ◽  
Cell Wall Biosynthesis ◽  
Bacterial Cells ◽  
E Coli ◽  
Essential Enzyme

ABSTRACT MurF is an essential enzyme of bacterial cell wall biosynthesis. Few MurF inhibitors have been reported, and none have displayed measurable antibacterial activity. Through the use of a MurF binding assay, a series of 8-hydroxyquinolines that bound to the Escherichia coli enzyme and inhibited its activity was identified. To derive additional chemotypes lacking 8-hydroxyquinoline, a known chelating moiety, a pharmacophore model was constructed from the series and used to select compounds for testing in the MurF binding and enzymatic inhibition assays. Whereas the original diverse library yielded 0.01% positive compounds in the binding assay, of which 6% inhibited MurF enzymatic activity, the pharmacophore-selected set yielded 14% positive compounds, of which 37% inhibited the enzyme, suggesting that the model enriched for compounds with affinity to MurF. A 4-phenylpiperidine (4-PP) derivative identified by this process displayed antibacterial activity (MIC of 8 μg/ml against permeable E. coli) including cell lysis and a 5-log10-unit decrease in CFU. Importantly, treatment of E. coli with 4-PP resulted in a 15-fold increase in the amount of the MurF UDP-MurNAc-tripeptide substrate, and a 50% reduction in the amount of the MurF UDP-MurNAc-pentapeptide product, consistent with inhibition of the MurF enzyme within bacterial cells. Thus, 4-PP is the first reported inhibitor of the MurF enzyme that may contribute to antibacterial activity by interfering with cell wall biosynthesis.

scholarly journals Bacterial Cell Enlargement Requires Control of Cell Wall Stiffness Mediated by Peptidoglycan Hydrolases

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Richard Wheeler ◽  
Robert D. Turner ◽  
Richard G. Bailey ◽  
Bartłomiej Salamaga ◽  
Stéphane Mesnage ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Fundamental Problem ◽  
Biochemical Properties ◽  
Major Advance ◽  
Bacterial Cells ◽  
Cell Enlargement ◽  
Content Type ◽  
Wall Stiffness ◽  
Peptidoglycan Hydrolases

ABSTRACTMost bacterial cells are enclosed in a single macromolecule of the cell wall polymer, peptidoglycan, which is required for shape determination and maintenance of viability, while peptidoglycan biosynthesis is an important antibiotic target. It is hypothesized that cellular enlargement requires regional expansion of the cell wall through coordinated insertion and hydrolysis of peptidoglycan. Here, a group of (apparent glucosaminidase) peptidoglycan hydrolases are identified that are together required for cell enlargement and correct cellular morphology ofStaphylococcus aureus, demonstrating the overall importance of this enzyme activity. These are Atl, SagA, ScaH, and SagB. The major advance here is the explanation of the observed morphological defects in terms of the mechanical and biochemical properties of peptidoglycan. It was shown that cells lacking groups of these hydrolases have increased surface stiffness and, in the absence of SagB, substantially increased glycan chain length. This indicates that, beyond their established roles (for example in cell separation), some hydrolases enable cellular enlargement by making peptidoglycan easier to stretch, providing the first direct evidence demonstrating that cellular enlargement occurs via modulation of the mechanical properties of peptidoglycan.IMPORTANCEUnderstanding bacterial growth and division is a fundamental problem, and knowledge in this area underlies the treatment of many infectious diseases. Almost all bacteria are surrounded by a macromolecule of peptidoglycan that encloses the cell and maintains shape, and bacterial cells must increase the size of this molecule in order to enlarge themselves. This requires not only the insertion of new peptidoglycan monomers, a process targeted by antibiotics, including penicillin, but also breakage of existing bonds, a potentially hazardous activity for the cell. UsingStaphylococcus aureus, we have identified a set of enzymes that are critical for cellular enlargement. We show that these enzymes are required for normal growth and define the mechanism through which cellular enlargement is accomplished, i.e., by breaking bonds in the peptidoglycan, which reduces the stiffness of the cell wall, enabling it to stretch and expand, a process that is likely to be fundamental to many bacteria.

scholarly journals Optical Tweezers Cause Physiological Damage to Escherichia coli and Listeria Bacteria

2008 ◽  
Vol 74 (8) ◽  
pp. 2441-2446 ◽  
Author(s):  
M. B. Rasmussen ◽  
L. B. Oddershede ◽  
H. Siegumfeldt
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Optical Tweezers ◽  
Laser Power ◽  
Fluorescent Protein ◽  
Growth Conditions ◽  
Ph Gradient ◽  
Bacterial Cells ◽  
E Coli

ABSTRACT We investigated the degree of physiological damage to bacterial cells caused by optical trapping using a 1,064-nm laser. The physiological condition of the cells was determined by their ability to maintain a pH gradient across the cell wall; healthy cells are able to maintain a pH gradient over the cell wall, whereas compromised cells are less efficient, thus giving rise to a diminished pH gradient. The pH gradient was measured by fluorescence ratio imaging microscopy by incorporating a pH-sensitive fluorescent probe, green fluorescent protein or 5(6)-carboxyfluorescein diacetate succinimidyl ester, inside the bacterial cells. We used the gram-negative species Escherichia coli and three gram-positive species, Listeria monocytogenes, Listeria innocua, and Bacillus subtilis. All cells exhibited some degree of physiological damage, but optically trapped E. coli and L. innocua cells and a subpopulation of L. monocytogenes cells, all grown with shaking, showed only a small decrease in pH gradient across the cell wall when trapped by 6 mW of laser power for 60 min. However, another subpopulation of Listeria monocytogenes cells exhibited signs of physiological damage even while trapped at 6 mW, as did B. subtilis cells. Increasing the laser power to 18 mW caused the pH gradient of both Listeria and E. coli cells to decrease within minutes. Moreover, both species of Listeria exhibited more-pronounced physiological damage when grown without shaking than was seen in cells grown with shaking, and the degree of damage is therefore also dependent on the growth conditions.

scholarly journals Humidity-Dependent Bacterial Cells Functional Morphometry Investigations Using Atomic Force Microscope

2010 ◽  
Vol 2010 ◽  
pp. 1-5 ◽  
Author(s):  
Hike Nikiyan ◽  
Alexey Vasilchenko ◽  
Dmitry Deryabin
Keyword(s):  
Cell Wall ◽  
Relative Humidity ◽  
Morphological Properties ◽  
Wall Structure ◽  
Bacterial Cells ◽  
Wall Roughness ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Wide Range

The effect of a relative humidity (RH) in a range of 93–65% on morphological and elastic properties ofBacillus cereusandEscherichia colicells was evaluated using atomic force microscopy. It is shown that gradual dehumidification of bacteria environment has no significant effect on cell dimensional features and considerably decreases them only at 65% RH. The increasing of the bacteria cell wall roughness and elasticity occurs at the same time. Observed changes indicate that morphological properties ofB. cereusare rather stable in wide range of relative humidity, whereasE. coliare more sensitive to drying, significantly increasing roughness and stiffness parameters at RH 84% RH. It is discussed the dependence of the response features on differences in cell wall structure of gram-positive and gram-negative bacterial cells.

scholarly journals An Exhaustive Multiple Knockout Approach to Understanding Cell Wall Hydrolase Function in Bacillus subtilis

2021 ◽  
Author(s):  
Sean Wilson ◽  
Ethan Garner
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Growth ◽  
Bond Cleavage ◽  
Normal Growth ◽  
Bacterial Cells ◽  
Single Strain ◽  
Cell Wall Hydrolase ◽  
Cell Wall Hydrolases ◽  
Synthetically Lethal

ABSTRACTMost bacteria are surrounded by their cell wall, a highly crosslinked protective envelope of peptidoglycan. To grow, bacteria must continuously remodel their wall, inserting new material and breaking old bonds. Bond cleavage is performed by cell wall hydrolases, allowing the wall to expand. Understanding the functions of individual hydrolases has been impeded by their redundancy: single knockouts usually present no phenotype. We used an exhaustive multiple-knockout approach to determine the minimal set of hydrolases required for growth in Bacillus subtilis. We identified 42 candidate cell wall hydrolases. Strikingly, we were able to remove all but two of these genes in a single strain; this “Δ40” strain shows a normal growth rate, indicating that none of the 40 hydrolases are necessary for cell growth. The Δ40 strain does not shed old cell wall, demonstrating that turnover is not essential for growth.The remaining two hydrolases in the Δ40 strain are LytE and CwlO, previously shown to be synthetically lethal. Either can be knocked out in Δ40, indicating that either hydrolase alone is sufficient for cell growth. Environmental screening and zymography revealed that LytE activity is inhibited by Mg2+ and that RlpA-like proteins may stimulate LytE activity. Together, these results demonstrate that the only essential function of cell wall hydrolases in B. subtilis is to enable cell growth by expanding the wall and that LytE or CwlO alone is sufficient for this function. These experiments introduce the Δ40 strain as a tool to study hydrolase activity and regulation in B. subtilis.IMPORTANCEIn order to grow, bacterial cells must both create and break down their cell wall. The enzymes that are responsible for these processes are the target of some of our best antibiotics. Our understanding of the proteins that break down the wall – cell wall hydrolases – has been limited by redundancy among the large number of hydrolases many bacteria contain. To solve this problem, we identified 42 cell wall hydrolases in Bacillus subtilis and created a strain lacking 40 of them. We show that cells can survive using only a single cell wall hydrolase; this means that to understand the growth of B. subtilis in standard laboratory conditions, it is only necessary to study a very limited number of proteins, simplifying the problem substantially. We additionally show that the Δ40 strain is a research tool to characterize hydrolases, using it to identify 3 ‘helper’ hydrolases that act in certain stress conditions.

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