scholarly journals The active repertoire of Escherichia coli peptidoglycan amidases varies with physiochemical environment

2020 ◽  
Author(s):  
Elizabeth A. Mueller ◽  
Abbygail G. Iken ◽  
Mehmet Ali Öztürk ◽  
Mirko Schmitz ◽  
Barbara Di Ventura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Cell Separation ◽  
Antimicrobial Agents ◽  
Model Organism ◽  
Growth Conditions ◽  
Low Ph ◽  
E Coli ◽  
Ph Dependent ◽  
Stimulation Of

ABSTRACTNearly all bacteria are encased in a peptidoglycan cell wall, an essential crosslinked matrix of polysaccharide strands and short peptide stems. In the Gram-negative model organism Escherichia coli, more than forty cell wall synthases and autolysins coordinate the growth and division of the peptidoglycan sacculus in the periplasm. The precise contribution of many of these enzymes to cell wall metabolism remains unclear due to significant apparent redundancy, particularly among the cell wall autolysins. E. coli produces three major LytC-type-N-acetylmuramoyl-L-alanine amidases, which share a role in separating the newly formed daughter cells during cytokinesis. Here, we reveal two of the three amidases exhibit growth medium-dependent changes in activity. Specifically, we report acidic growth conditions stimulate AmiB—and to a lesser extent, AmiC—activity. Combining computational and genetic analysis, we demonstrate that low pH-dependent stimulation of AmiB requires three periplasmic amidase activators: EnvC, NlpD, and YgeR. Altogether, our findings support overlapping, but not redundant, roles for the E. coli amidases in cell separation and illuminate the physiochemical environment as an important mediator of cell wall enzyme activity.IMPORTANCEPenicillin and related β-lactam antibiotics targeting the bacterial cell wall synthesis are among the most commonly prescribed antimicrobials worldwide. However, rising rates of antibiotic resistance and tolerance jeopardize their continued clinical use. Development of new cell wall active therapeutics, including those targeting cell wall autolysins, has been stymied in part due to high levels of apparent enzymatic redundancy. In this study, we report a subset of E. coli amidases involved in cell separation during cell division are not redundant and instead are preferentially active during growth in distinct pH environments. Specifically, we discover E. coli amidases AmiB and AmiC are activated by acidic pH. Three semi-redundant periplasmic regulators—NlpD, EnvC, and YgeR—collectively mediate low pH-dependent stimulation of amidase activity. This discovery contributes to our understanding of how the cell wall remains robust across diverse environmental conditions and reveals opportunities for the development of condition-specific antimicrobial agents.

scholarly journals The Redundancy of Peptidoglycan Carboxypeptidases Ensures Robust Cell Shape Maintenance inEscherichia coli

mBio ◽  
2016 ◽  
Vol 7 (3) ◽  
Author(s):  
Katharina Peters ◽  
Suresh Kannan ◽  
Vincenzo A. Rao ◽  
Jacob Biboy ◽  
Daniela Vollmer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Shape ◽  
Normal Growth ◽  
Local Environment ◽  
Growth Conditions ◽  
Low Ph ◽  
Acidic Ph ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Morphological Defects

ABSTRACTPeptidoglycan (PG) is an essential structural component of the bacterial cell wall and maintains the integrity and shape of the cell by forming a continuous layer around the cytoplasmic membrane. The thin PG layer ofEscherichia coliresides in the periplasm, a unique compartment whose composition and pH can vary depending on the local environment of the cell. Hence, the growth of the PG layer must be sufficiently robust to allow cell growth and division under different conditions. We have analyzed the PG composition of 28 mutants lacking multiple PG enzymes (penicillin-binding proteins [PBPs]) after growth in acidic or near-neutral-pH media. Statistical analysis of the muropeptide profiles identifieddd-carboxypeptidases (DD-CPases) that were more active in cells grown at acidic pH. In particular, the absence of the DD-CPase PBP6b caused a significant increase in the pentapeptide content of PG as well as morphological defects when the cells were grown at acidic pH. Other DD-CPases (PBP4, PBP4b, PBP5, PBP6a, PBP7, and AmpH) and the PG synthase PBP1B made a smaller or null contribution to the pentapeptide-trimming activity at acidic pH. We solved the crystal structure of PBP6b and also demonstrated that the enzyme is more stable and has a lowerKmat acidic pH, explaining why PBP6b is more active at low pH. Hence, PBP6b is a specialized DD-CPase that contributes to cell shape maintenance at low pH, andE. coliappears to utilize redundant DD-CPases for normal growth under different conditions.IMPORTANCEEscherichia colirequires peptidoglycandd-carboxypeptidases to maintain cell shape by controlling the amount of pentapeptide substrates available to the peptidoglycan synthetic transpeptidases. WhyE. colihas eight, seemingly redundantdd-carboxypeptidases has remained unknown. We now show that one of thesedd-carboxypeptidases, PBP6b, is important for cell shape maintenance in acidic growth medium, consistent with the higher activity and stability of the enzyme at low pH. Hence, the presence of multipledd-carboxypeptidases with different enzymatic properties may allowE. colito maintain a normal cell shape under various growth conditions.

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 Regulation of FtsZ levels inEscherichia coliin slow growth conditions

2018 ◽  
Author(s):  
Jaana Männik ◽  
Bryant E. Walker ◽  
Jaan Männik
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Cell Division ◽  
Slow Growth ◽  
Model Organism ◽  
Growth Conditions ◽  
Rapid Degradation ◽  
E Coli ◽  
Z Ring ◽  
Cell Envelopes

AbstractA key regulator of cell division in most walled bacteria is the FtsZ protein that assembles into protofilaments attached to the membrane at midcell. These dynamic protofilament assemblies, known as the Z-ring, act as a scaffold for more than two dozen proteins involved in synthesis of septal cell envelopes. What triggers the formation of the Z-ring during the cell cycle is poorly understood. InEscherichia colimodel organism, the common view is that FtsZ concentration is constant throughout its doubling time and therefore regulation of assembly should be controlled by some yet to be identified protein-protein interactions. Here we show using quantitative analysis of newly developed fluorescent reporter that FtsZ concentration varies in a cell-cycle dependent manner in slow growth conditions and that upregulation of FtsZ synthesis correlates with the formation of the Z-ring. About 4-fold upregulation of FtsZ synthesis in the first half of the cell cycle is followed by its rapid degradation by ClpXP protease in the last 10% of the cell cycle. The initiation of rapid degradation coincides with dissociation of FtsZ from the septum. Altogether, our data indicate that the Z-ring formation in slow growth conditions inE. coliis controlled by a regulatory sequence where upregulation of an essential cell cycle factor is followed by its degradation.SignificanceFtsZ is the key regulator for bacterial cell division. It initiates division by forming a dynamic ring-like structure, the Z-ring, at the mid-cell. Here we show that, contrarily to the current paradigm, FtsZ concentration inEscherichia colimodel organism varies throughout cell cycle in slow growth conditions. Faster FtsZ synthesis in the first half of the cell cycle is followed by its rapid degradation by ClpXP protease in the end of the cell cycle. Upregulation of FtsZ synthesis correlates with the formation of the Z-ring. Our data demonstrates that in slow growthE. colicell division progresses according to paradigm where upregulation of essential cell cycle factor is followed by its degradation.

scholarly journals New Insights into the Antimicrobial Action of Cinnamaldehyde towards Escherichia coli and Its Effects on Intestinal Colonization of Mice

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 302
Author(s):  
Wellison A. Pereira ◽  
Carlos Drielson S. Pereira ◽  
Raíssa G. Assunção ◽  
Iandeyara Savanna C. da Silva ◽  
Fabrícia S. Rego ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Antimicrobial Agents ◽  
Coli Strain ◽  
Antimicrobial Action ◽  
Wall Structure ◽  
Bacterial Disease ◽  
Intestinal Colonization ◽  
Adhesive Properties ◽  
E Coli

Escherichia coli is responsible for cases of diarrhea around the world, and some studies have shown the benefits of cinnamaldehyde in the treatment of bacterial disease. Therefore, the objective of this study was to evaluate the effects of cinnamaldehyde in mice colonized by pathogenic E. coli, as well as to provide more insights into its antimicrobial action mechanism. After determination of minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations, the interference of cinnamaldehyde in macromolecular pathways (synthesis of DNA, RNA, protein, and cell wall) was measured by incorporation of radioisotopes. The anti-adhesive properties of cinnamaldehyde towards E. coli 042 were evaluated using human epithelial type 2 (HEp-2) cells. Intestinal colonization was tested on mice, and the effect of cinnamaldehyde on Tenebrio molitor larvae. Cinnamaldehyde showed MIC and MBC values of 780 μg/mL and 1560 μg/mL, respectively; reduced the adhesion of E. coli 042 on HEp-2 cells; and affected all the synthetic pathways evaluated, suggesting that compost impairs the membrane/cell wall structure leading bacteria to total collapse. No effect on the expression of genes related to the SOS pathway (sulA and dinB1) was observed. The compound did not interfere with cell viability and was not toxic against T. molitor larvae. In addition, cinnamaldehyde-treated mice exhibited lower levels of colonization by E. coli 042 than the untreated group. Therefore, the results show that cinnamaldehyde is effective in treating the pathogenic E. coli strain 042 and confirm it as a promising lead molecule for the development of antimicrobial agents.

Sites of Adsorption of the Bacteriophages T3 and T5 on the Wall of Escherichia Coli B.

1967 ◽  
Vol 25 ◽  
pp. 96-97
Author(s):  
Manfred E. Bayer
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Electron Microscope ◽  
Uranyl Acetate ◽  
E Coli ◽  
Receptor Sites ◽  
Rigid Cell Wall ◽  
Host Cell Surface ◽  
Bacterial Viruses ◽  
Escherichia Coli B

Bacterial viruses adsorb specifically to receptors on the host cell surface. Although the chemical composition of some of the cell wall receptors for bacteriophages of the T-series has been described and the number of receptor sites has been estimated to be 150 to 300 per E. coli cell, the localization of the sites on the bacterial wall has been unknown.When logarithmically growing cells of E. coli are transferred into a medium containing 20% sucrose, the cells plasmolize: the protoplast shrinks and becomes separated from the somewhat rigid cell wall. When these cells are fixed in 8% Formaldehyde, post-fixed in OsO4/uranyl acetate, embedded in Vestopal W, then cut in an ultramicrotome and observed with the electron microscope, the separation of protoplast and wall becomes clearly visible, (Fig. 1, 2). At a number of locations however, the protoplasmic membrane adheres to the wall even under the considerable pull of the shrinking protoplast. Thus numerous connecting bridges are maintained between protoplast and cell wall. Estimations of the total number of such wall/membrane associations yield a number of about 300 per cell.

Antimicrobial Effects of Zataria multiflora and Ocimum basilicum on Escherichia coli O157:H7 During Ripening of Traditional Lighvan Cheese

2020 ◽  
Vol 16 (3) ◽  
pp. 373-380
Author(s):  
Mohammad B. Zendeh ◽  
Vadood Razavilar ◽  
Hamid Mirzaei ◽  
Khosrow Mohammadi
Keyword(s):  
Escherichia Coli ◽  
Essential Oils ◽  
Dairy Products ◽  
Antimicrobial Agents ◽  
Escherichia Coli O157 ◽  
Zataria Multiflora ◽  
Antimicrobial Effects ◽  
E Coli ◽  
Common Causes ◽  
Lighvan Cheese

Background: Escherichia coli O157:H7 is one of the most common causes of contamination in Lighvan cheese processing. Using from natural antimicrobial essential oils is applied method to decrease the rate of microbial contamination of dairy products. The present investigation was done to study the antimicrobial effects of Z. multiflora and O. basilicum essential oils on survival of E. coli O157:H7 during ripening of traditional Lighvan cheese. Methods: Leaves of the Z. multiflora and O. basilicum plants were subjected to the Clevenger apparatus. Concentrations of 0, 100 and 200 ppm of the Z. multiflora and 0, 50 and 100 ppm of O. basilicum essential oils and also 103 and 105 cfu/ml numbers of E. coli O157:H7 were used. The numbers of the E. coli O157:H7 bacteria were analyzed during the days 0, 30, 60 and 90 of the ripening period. Results: Z. multiflora and O. basilicum essential oils had considerable antimicrobial effects against E. coli O157:H7. Using the essential oils caused decrease in the numbers of E. coli O157:H7 bacteria in 90th days of ripening (P <0.05). Using from Z. multiflora at concentration of 200 ppm can reduce the survival of E. coli O157:H7 in Lighvan cheese. Conclusion: Using Z. multiflora and O. basilicum essential oils as good antimicrobial agents can reduce the risk of foodborne bacteria and especially E. coli O157:H7 in food products.

scholarly journals A Rhodobacter sphaeroides Protein Mechanistically Similar to Escherichia coli DksA Regulates Photosynthetic Growth

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Christopher W. Lennon ◽  
Kimberly C. Lemmer ◽  
Jessica L. Irons ◽  
Max I. Sellman ◽  
Timothy J. Donohue ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Structure Function ◽  
Rhodobacter Sphaeroides ◽  
Fatty Acid Content ◽  
Regulatory Protein ◽  
Growth Conditions ◽  
Globular Domain ◽  
Content Type ◽  
E Coli

ABSTRACTDksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the “stringent response” to nutrient starvation in the gammaproteobacteriumEscherichia coliand for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly relatedRhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length toE. coliDksA but lacks the Zn finger motif of theE. coliDksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of anE. colistrain lacking thedksAgene and modulates transcriptionin vitrowithE. coliRNA polymerase (RNAP) similarly toE. coliDksA. RSP2654 reduces RNAP-promoter complex stabilityin vitrowith RNAPs fromE. coliorR. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity toE. coliDksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsphas distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp.IMPORTANCEThe role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis inRhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

scholarly journals Impact of Feed Supplementation with Antimicrobial Agents on Growth Performance of Broiler Chickens, Clostridium perfringens and Enterococcus Counts, and Antibiotic Resistance Phenotypes and Distribution of Antimicrobial Resistance Determinants in Escherichia coli Isolates

2007 ◽  
Vol 73 (20) ◽  
pp. 6566-6576 ◽  
Author(s):  
Moussa S. Diarra ◽  
Fred G. Silversides ◽  
Fatoumata Diarrassouba ◽  
Jane Pritchard ◽  
Luke Masson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Antimicrobial Resistance ◽  
Broiler Chickens ◽  
Antimicrobial Agents ◽  
Growth Promoters ◽  
Feed Supplementation ◽  
E Coli ◽  
Resistance Determinants ◽  
Class 1

ABSTRACT The effects of feed supplementation with the approved antimicrobial agents bambermycin, penicillin, salinomycin, and bacitracin or a combination of salinomycin plus bacitracin were evaluated for the incidence and distribution of antibiotic resistance in 197 commensal Escherichia coli isolates from broiler chickens over 35 days. All isolates showed some degree of multiple antibiotic resistance. Resistance to tetracycline (68.5%), amoxicillin (61.4%), ceftiofur (51.3%), spectinomycin (47.2%), and sulfonamides (42%) was most frequent. The levels of resistance to streptomycin, chloramphenicol, and gentamicin were 33.5, 35.5, and 25.3%, respectively. The overall resistance levels decreased from day 7 to day 35 (P < 0.001). Comparing treatments, the levels of resistance to ceftiofur, spectinomycin, and gentamicin (except for resistance to bacitracin treatment) were significantly higher in isolates from chickens receiving feed supplemented with salinomycin than from the other feeds (P < 0.001). Using a DNA microarray analysis capable of detecting commonly found antimicrobial resistance genes, we characterized 104 tetracycline-resistant E. coli isolates from 7- to 28-day-old chickens fed different growth promoters. Results showed a decrease in the incidence of isolates harboring tet(B), bla TEM, sulI, and aadA and class 1 integron from days 7 to 35 (P < 0.01). Of the 84 tetracycline-ceftiofur-resistant E. coli isolates, 76 (90.5%) were positive for bla CMY-2. The proportions of isolates positive for sulI, aadA, and integron class 1 were significantly higher in salinomycin-treated chickens than in the control or other treatment groups (P < 0.05). These data demonstrate that multiantibiotic-resistant E. coli isolates can be found in broiler chickens regardless of the antimicrobial growth promoters used. However, the phenotype and the distribution of resistance determinants in E. coli can be modulated by feed supplementation with some of the antimicrobial agents used in broiler chicken production.

scholarly journals Occurrence of toxigenic Escherichia coli in raw milk cheese in Brazil

2007 ◽  
Vol 59 (2) ◽  
pp. 508-512 ◽  
Author(s):  
B.R. Paneto ◽  
R.P. Schocken-Iturrino ◽  
C. Macedo ◽  
E. Santo ◽  
J.M. Marin
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Antimicrobial Agents ◽  
Raw Milk ◽  
Diffusion Method ◽  
Chain Reaction ◽  
Disk Diffusion Method ◽  
E Coli ◽  
Polymerase Chain ◽  
Raw Milk Cheese

The occurrence of toxigenic Escherichia coli in raw milk cheese was surveyed in Middle Western Brazil. Fifty samples of cheese from different supermarkets were analyzed for E.coli. The isolates were serotyped and screened for the presence of verotoxigenic E. coli (VTEC) and enterotoxigenic E. coli (ETEC) by Polymerase Chain Reaction (PCR). The susceptibility to thirteen antimicrobial agents was evaluated by the disk diffusion method. E.coli were recovered from 48 (96.0%) of the samples. The serogroups identified were O125 (6.0%), O111 (4.0%), O55 (2.0%) and O119 (2.0%). Three (6.0%) and 1(2.0%) of the E.coli isolates were VTEC and ETEC, respectively. Most frequent resistance was observed to the following antimicrobials: cephalothin (60.0%), nalidixic acid (40.0%), doxycyclin (33.0%), tetracycline (31.0%) and ampicillin (29.0%).

scholarly journals pH-Dependent Catabolic Protein Expression during Anaerobic Growth of Escherichia coli K-12

2004 ◽  
Vol 186 (1) ◽  
pp. 192-199 ◽  
Author(s):  
Elizabeth Yohannes ◽  
D. Michael Barnhart ◽  
Joan L. Slonczewski
Keyword(s):  
Escherichia Coli ◽  
Ph Regulation ◽  
Metabolic Enzymes ◽  
Anaerobic Growth ◽  
High Ph ◽  
E Coli ◽  
Catabolic Enzymes ◽  
Periplasmic Proteins ◽  
Ph Dependent ◽  
K 12

ABSTRACT During aerobic growth of Escherichia coli, expression of catabolic enzymes and envelope and periplasmic proteins is regulated by pH. Additional modes of pH regulation were revealed under anaerobiosis. E. coli K-12 strain W3110 was cultured anaerobically in broth medium buffered at pH 5.5 or 8.5 for protein identification on proteomic two-dimensional gels. A total of 32 proteins from anaerobic cultures show pH-dependent expression, and only four of these proteins (DsbA, TnaA, GatY, and HdeA) showed pH regulation in aerated cultures. The levels of 19 proteins were elevated at the high pH; these proteins included metabolic enzymes (DhaKLM, GapA, TnaA, HisC, and HisD), periplasmic proteins (ProX, OppA, DegQ, MalB, and MglB), and stress proteins (DsbA, Tig, and UspA). High-pH induction of the glycolytic enzymes DhaKLM and GapA suggested that there was increased fermentation to acids, which helped neutralize alkalinity. Reporter lac fusion constructs showed base induction of sdaA encoding serine deaminase under anaerobiosis; in addition, the glutamate decarboxylase genes gadA and gadB were induced at the high pH anaerobically but not with aeration. This result is consistent with the hypothesis that there is a connection between the gad system and GabT metabolism of 4-aminobutanoate. On the other hand, 13 other proteins were induced by acid; these proteins included metabolic enzymes (GatY and AckA), periplasmic proteins (TolC, HdeA, and OmpA), and redox enzymes (GuaB, HmpA, and Lpd). The acid induction of NikA (nickel transporter) is of interest because E. coli requires nickel for anaerobic fermentation. The position of the NikA spot coincided with the position of a small unidentified spot whose induction in aerobic cultures was reported previously; thus, NikA appeared to be induced slightly by acid during aeration but showed stronger induction under anaerobic conditions. Overall, anaerobic growth revealed several more pH-regulated proteins; in particular, anaerobiosis enabled induction of several additional catabolic enzymes and sugar transporters at the high pH, at which production of fermentation acids may be advantageous for the cell.

Sign in / Sign up

Export Citation Format

Share Document