scholarly journals Colicin-mediated transport of DNA through the iron transporter FepA

2021 ◽  
Author(s):  
Ruth Cohen-Khait ◽  
Ameya Harmalkar ◽  
Phuong Pham ◽  
Melissa N Webby ◽  
Nicholas G Housden ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Active Transport ◽  
Large Scale ◽  
Receptor Interaction ◽  
Cellular Receptor ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Colicin B

Colicins are protein antibiotics used by bacteria to eliminate competing Escherichia coli. A key event in the selective colicin function is a highly specific initial recognition step of an outer membrane (OM) receptor, which consequently allows the active transport of the colicin across the otherwise impervious OM. Though the colicin-receptor interaction is exclusive, the translocation process is likely to be universal as many receptors and colicins have surprisingly simi-lar 3D folds. Here, using a combination of photo-activated crosslinking, mass spectrometry, and structural modeling, we reveal how colicin B (ColB) associates with its OM receptor FepA. We demonstrate that complex formation is co-incident with a large-scale conformational change in the colicin. In vivo crosslinking experiments and further simula-tions of the translocation process indicate that part of the colicin engages active transport by disguising itself to part of the cellular receptor. Applying live-cell fluorescence imaging we were able to follow ColB into E. coli and localize it within the periplasm. Finally, we demonstrate that single-stranded DNA coupled to ColB is transported into the bacte-rial periplasm, emphasizing that the import routes of colicins can be exploited to carry large cargo molecules into Gram-negative bacteria.

scholarly journals Inhibition of Multidrug-Resistant Gram-Positive and Gram-Negative Bacteria by a Photoactivated Porphyrin

2017 ◽  
Vol 66 (4) ◽  
pp. 533-536 ◽  
Author(s):  
Moreno Bondi ◽  
Anna Mazzini ◽  
Simona de Niederhäusern ◽  
Ramona Iseppi ◽  
Patrizia Messi
Keyword(s):  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Gram Negative Bacteria ◽  
Multidrug Resistant Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
E Coli ◽  
In Vitro Antibacterial Activity

The authors studied the in vitro antibacterial activity of the photo-activated porphyrin meso-tri(N-methyl-pyridyl), mono(N-tetradecyl-pyridyl)porphine (C14) against four multidrug-resistant bacteria: Staphylococcus aureus, Enterococcus faecalis (Gram-positive), Escherichia coli, Pseudomonas aeruginosa (Gram-negative). Using 10 μg/ml of porphyrin and 60 sec irradiation we observed the remarkable susceptibility of S. aureus and E. faecalis to treatment while, under the same conditions, E. coli and P. aeruginosa showed very low susceptibility. In a later stage, suspensions of Gram-negative bacteria were processed with EDTA before photo-activation, obtaining a significant decrease in viable counts. In view of the results, if the combination of low porphyrin concentrations and short irradiation times will be effective in vivo also, this approach could be a possible alternative to antibiotics, in particular against localized infections due to multidrug-resistant microorganisms.

scholarly journals Digitalizing heterologous gene expression in Gram-negative bacteria with a portable on/off module

2019 ◽  
Author(s):  
Belén Calles ◽  
Angel Goñi-Moreno ◽  
Víctor de Lorenzo
Keyword(s):  
Heterologous Gene Expression ◽  
Host Bacterium ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Inducible Promoters ◽  
E Coli ◽  
Naturally Occurring ◽  
Conditional Expression ◽  
Colicin E3

ABSTRACTWhile prokaryotic promoters controlled by signal-responding regulators typically display a range of input/output ratios when exposed to cognate inducers, virtually no naturally occurring cases are known to have an off state of zero transcription—as ideally needed for synthetic circuits. To overcome this problem we have modelled and implemented simple digitalizer module that completely suppresses the basal level of otherwise strong promoters in such a way that expression in the absence of induction is entirely impeded. The circuit involves the interplay of a translation-inhibitory sRNA with the translational coupling of the gene of interest to a repressor such as LacI. The digitalizer module was validated with the strong inducible promoters Pm (induced by XylS in the presence of benzoate) and PalkB (induced by AlkS/dicyclopropylketone) and shown to perform effectively both in E. coli and the soil bacterium Pseudomonas putida. The distinct expression architecture allowed cloning and conditional expression of e.g. colicin E3, one molecule of which per cell suffices to kill the host bacterium. Revertants that escaped ColE3 killing were not found in hosts devoid of insertion sequences, suggesting that mobile elements are a major source of circuit inactivation in vivo.

scholarly journals In vivo targeting of E. coli with vancomycin-arginine

2021 ◽  
Author(s):  
Lewis F. Neville ◽  
Itamar Shalit ◽  
Peter A. Warn ◽  
Marc H. Scheetz ◽  
Jiuzhi Sun ◽  
...  
Keyword(s):  
Low Frequency ◽  
Muscle Model ◽  
Thigh Muscle ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Further Development

The ability of vancomycin-arginine (V-r) to extend the spectrum of activity of glycopeptides to gram-negative bacteria was investigated. Its MIC towards E. coli including β-lactamase expressing Ambler classes A, B, and D was 8-16μg/ml. Addition of 8×MIC V-r to E. coli was acutely bactericidal and associated with a low frequency of resistance (< 2.3×10−10). In vivo, V-r markedly reduced E. coli burden by >7 log10 CFU/g in a thigh muscle model. These data warrant further development of V-r in combatting E. coli, including resistant forms.

scholarly journals Spectroscopic, Thermal, Electrochemical, and Antimicrobial Studies of Mononuclear Manganese(II) Ditolyldithiophosphates

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
Ruchi Khajuria ◽  
Atiya Syed ◽  
Sandeep Kumar ◽  
Sushil K. Pandey
Keyword(s):  
Mass Spectrometry ◽  
Cyclic Voltammetry ◽  
Magnetic Susceptibility ◽  
Molar Conductance ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Antimicrobial Studies ◽  
Esi Mass Spectrometry ◽  
Uv Visible

New complexes of manganese(II) corresponding to [{(ArO)2PS2}2Mn] and [{(ArO)2PS2}2Mn.nL] (Ar=o-,m-,p-CH3C6H4andp-Cl-m-CH3C6H3;n=1, L = N2C12H8, N2C10H8;n=2, L = NC5H5, P(C6H5)3) have been synthesized and characterized by microelemental analyses (C, H, and N), magnetic susceptibility, molar conductance, thermogravimetric, cyclic voltammetry, and spectral analyses including ESI mass spectrometry, IR, and UV-visible. The presence of a four-and-six coordinated Mn atoms has been established in the complexes and adducts, respectively. Antimicrobial screening of the complexes against gram negative bacteriaE. coli,K. pneumonia,andP. aeruginosaand fungusS. rolfsiihas shown potential bioactivity.

scholarly journals The outer membrane proteins OmpA, FhuA, OmpF, EstA, BtuB and OmpX have unique lipopolysaccharide fingerprints

2018 ◽  
Author(s):  
Jonathan Shearer ◽  
Damien Jefferies ◽  
Syma Khalid
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Coarse Grain ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Unique Pattern ◽  
E Coli ◽  
Outer Leaflet ◽  
Wide Range

AbstractThe outer membrane of Gram-negative bacteria has a highly complex asymmetrical architecture, containing a mixture of phospholipids in the inner leaflet and in the outer leaflet they contain almost exclusively lipopolysaccharide (LPS) molecules. In E. coli, the outer membrane contains a wide range proteins with a beta barrel architecture, that vary in size from the smallest having eight strands to larger barrels composed of twenty-two strands. Here we report coarse-grain molecular dynamics simulations of six proteins from the E. coli outer membrane OmpA, OmpX, BtuB, FhuA, OmpF and EstA in a range of membrane environments, which are representative of the in vivo for different strains of E. coli. We show that each protein has a unique pattern of interaction with the surrounding membrane, which is influenced by the composition of the protein, the level of LPS in the outer leaflet and the differing mobilities of the lipids in the two leaflets of the membrane. Overall we present analyses from over 200 microseconds of simulation for each protein.Author summaryWe present data from over 200 microseconds of coarse-grain simulations that show the complexities of protein-lipid interactions within the outer membranes of Gram-negative bacteria. We show that the slow movement of lipolysaccharide molecules necessitate simulations of over 30 microsecond duration to achieve converged properties such as protein tilt angle. Each of the six proteins studied here shows a unique pattern of interactions with the outer membrane and thus constitute a ‘fingerprint’ or ‘signature’.

scholarly journals In VitroEfficacy of Nonantibiotic Treatments on Biofilm Disruption of Gram-Negative Pathogens and anIn VivoModel of Infectious Endometritis Utilizing Isolates from the Equine Uterus

2015 ◽  
Vol 54 (3) ◽  
pp. 631-639 ◽  
Author(s):  
Ryan A. Ferris ◽  
Patrick M. McCue ◽  
Grace I. Borlee ◽  
Kristen D. Loncar ◽  
Margo L. Hennet ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Biofilm Disruption ◽  
Infectious Endometritis ◽  
Clinical Cases

In this study, we evaluated the ability of the equine clinical treatmentsN-acetylcysteine, EDTA, and hydrogen peroxide to disruptin vitrobiofilms and kill equine reproductive pathogens (Escherichia coli,Pseudomonas aeruginosa, orKlebsiella pneumoniae) isolated from clinical cases.N-acetylcysteine (3.3%) decreased biofilm biomass and killed bacteria within the biofilms ofE. coliisolates. The CFU of recoverableP. aeruginosaandK. pneumoniaeisolates were decreased, but the biofilm biomass was unchanged. Exposure to hydrogen peroxide (1%) decreased the biofilm biomass and reduced the CFU ofE. coliisolates,K. pneumoniaeisolates were observed to have a reduction in CFU, and minimal effects were observed forP. aeruginosaisolates. Chelating agents (EDTA formulations) reducedE. coliCFU but were ineffective at disrupting preformed biofilms or decreasing the CFU ofP. aeruginosaandK. pneumoniaewithin a biofilm. No single nonantibiotic treatment commonly used in equine veterinary practice was able to reduce the CFU and biofilm biomass of all three Gram-negative species of bacteria evaluated. Anin vivoequine model of infectious endometritis was also developed to monitor biofilm formation, utilizing bioluminescence imaging with equineP. aeruginosaisolates from this study. Following infection, the endometrial surface contained focal areas of bacterial growth encased in a strongly adherent “biofilm-like” matrix, suggesting that biofilms are present during clinical cases of infectious equine endometritis. Our results indicate that Gram-negative bacteria isolated from the equine uterus are capable of producing a biofilmin vitro, andP. aeruginosais capable of producing biofilm-like materialin vivo.

Immunogold labeling of CMP-KDO synthetase produced by recombinant E. Coli cells

1987 ◽  
Vol 45 ◽  
pp. 924-925
Author(s):  
F. A. Durum ◽  
R. G. Goldman ◽  
T. J. Bolling ◽  
M. F. Miller
Keyword(s):  
Inclusion Bodies ◽  
Large Scale ◽  
Recombinant Dna ◽  
Test System ◽  
Cell Protein ◽  
Gram Negative Bacteria ◽  
Cytoplasmic Inclusions ◽  
Isolation And Purification ◽  
E Coli ◽  
Low Concentrations

CMP-KDO synthetase (CKS) is an enzyme which plays a key role in the synthesis of LPS, an outer membrane component unique to gram negative bacteria. CKS activates KDO to CMP-KDO for incorporation into LPS. The enzyme is normally present in low concentrations (0.02% of total cell protein) which makes it difficult to perform large scale isolation and purification. Recently, the gene for CKS from E. coli was cloned and various recombinant DNA constructs overproducing CKS several thousandfold (unpublished data) were derived. Interestingly, no cytoplasmic inclusions of overproduced CKS were observed by EM (Fig. 1) which is in contrast to other reports of large proteinaceous inclusion bodies in various overproducing recombinant strains. The present immunocytochemical study was undertaken to localize CKS in these cells.Immune labeling conditions were first optimized using a previously described cell-free test system. Briefly, this involves soaking small blocks of polymerized bovine serum albumin in purified CKS antigen and subjecting them to various fixation, embedding and immunochemical conditions.

Disinfection of Fruits with Activated Water

2019 ◽  
Vol 10 ◽  
pp. 1864-1872
Author(s):  
Prof. Teodora P. Popova
Keyword(s):  
Antimicrobial Activity ◽  
Aqueous Solutions ◽  
Antimicrobial Properties ◽  
The Other ◽  
Gram Negative Bacteria ◽  
High Antimicrobial Activity ◽  
Gram Negative ◽  
E Coli ◽  
Number Of Microorganisms ◽  
Lower Effect

The effect of ionized aqueous solutions (anolytes and catholyte) in the processing of fruits (cherries, morellos, and strawberries) for decontamination has been tested. Freshly prepared analytes and catholyte without the addition of salts were used, as well as stored for 7 months anolytes, prepared with 0.5% NaCl and a combination of 0.5% NaCl and 0.5% Na2CO3. The anolyte prepared with a combination of 0.5% NaCl and 0.5% Na2CO3, as well as the anolyte obtained with 0.5% NaCl, exhibit high antimicrobial activity against the surface microflora of strawberries, cherries, and sour cherries. They inactivate E. coli for 15 minutes. The other species of the fam. Enterobacteriaceae were also affected to the maximum extent, as is the total number of microorganisms, especially in cherries and sour cherries. Even stored for 7 months, they largely retain their antimicrobial properties. Anolyte and catholyte, obtained without the addition of salts, showed a lower effect on the total number of microorganisms, but had a significant effect on Gram-negative bacteria, and especially with regard to the sanitary indicative E. coli.

Structure-Activity Relationship and Antimicrobial Evaluation of N-Phenylpyrazole Curcumin Derivatives

2020 ◽  
Vol 16 (4) ◽  
pp. 481-488
Author(s):  
Heli Sanghvi ◽  
Satyendra Mishra
Keyword(s):  
Antibacterial Activity ◽  
Gram Negative Bacteria ◽  
Pyrazole Derivatives ◽  
Gram Positive ◽  
Gram Negative ◽  
E Coli ◽  
Curcumin Derivatives ◽  
Structure Activity ◽  
Electron Donating Groups ◽  
Derivatives Of

Background: Curcumin, one of the most important pharmacologically significant natural products, has gained significant consideration among scientists for decades since its multipharmacological activities. 1, 3-Dicarbonyl moiety of curcumin was found to be accountable for the rapid degradation of curcumin molecule. The aim of present work is to replace 1, 3-dicarbonyl moiety of curcumin by pyrazole and phenylpyrazole derivatives with a view to improving its stability and to investigate the role of substitution in N-phenylpyrazole curcumin on its antibacterial activity against both Gram-positive as well as Gram-negative bacteria. Methods: Pyrazole derivatives of curcumin were prepared by heating curcumin with phenyhydrazine/ substituted phenyhydrazine derivatives in AcOH. The residue was purified by silica gel column chromatography. Structures of purified compounds were confirmed by 1H NMR and Mass spectroscopy. The synthesized compounds were evaluated for their antibacterial activity by the microdilution broth susceptibility test method against gram positive (S. aureus) and gram negative (E. coli). Results: Effects of substitution in N-phenylpyrazole curcumin derivatives against S. aureus and E. coli were studied. The most active N-(3-Nitrophenylpyrazole) curcumin (12) exhibits twenty-fold more potency against S. aureus (MIC: 10μg/mL)) and N-(2-Fluoroophenylpyrazole) curcumin (5) fivefold more potency against E. coli (MIC; 50 μg/mL) than N-phenylpyrazole curcumin (4). Whereas, a remarkable decline in anti-bacterial activity against S. aureus and E. coli was observed when electron donating groups were incorporated in N-phenylpyrazole curcumin (4). Comparative studies of synthesized compounds suggest the effects of electron withdrawing and electron donating groups on unsubstituted phenylpyrazole curcumin (4). Conclusion: The structure-activity relationship (SAR) results indicated that the electron withdrawing and electron donating at N-phenylpyrazole curcumin played key roles for their bacterial inhibitory effects. The results of the antibacterial evaluation showed that the synthesized pyrazole derivatives of curcumin displayed moderate to very high activity in S. aureus. In conclusion, the series of novel curcumin derivatives were designed, synthesized and tested for their antibacterial activities against S. aureus and E. coli. Among them, N-(3-Nitrophenylpyrazole curcumin; 12) was most active against S. aureus (Gram-positive) and N-(2-Fluoroophenylpyrazole) curcumin (5) against E. coli (Gram-negative) bacteria.

Quality Improvement of the DNA extracted by boiling method in Gram negative bacteria

2017 ◽  
Vol 6 (04) ◽  
pp. 5347 ◽  
Author(s):  
Omar B. Ahmed* ◽  
Anas S. Dablool
Keyword(s):  
Dna Extraction ◽  
Control Method ◽  
Extraction Procedure ◽  
Cost Effective ◽  
High Yield ◽  
Gram Negative Bacteria ◽  
Bacterial Dna ◽  
Gram Negative ◽  
E Coli

Several methods of Deoxyribonucleic acid (DNA) extraction have been applied to extract bacterial DNA. The amount and the quality of the DNA obtained for each one of those methods are variable. The study aimed to evaluate bacterial DNA extraction using conventional boiling method followed by alcohol precipitation. DNA extraction from Gram negative bacilli was extracted and precipitated using boiling method with further precipitation by ethanol. The extraction procedure performed using the boiling method resulted in high DNA yields for both E. coli and K. pneumoniae bacteria in (199.7 and 285.7μg/ml, respectively) which was close to control method (229.3 and 440.3μg/ml). It was concluded that after alcohol precipitation boiling procedure was easy, cost-effective, and applicable for high-yield quality of DNA in Gram-negative bacteria.

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