scholarly journals Stabilization of pSW100 from Pantoea stewartii by the F Conjugation System

2008 ◽  
Vol 190 (10) ◽  
pp. 3681-3689 ◽  
Author(s):  
Mei-Hui Lin ◽  
Shih-Tung Liu
Keyword(s):  
Escherichia Coli ◽  
Genetic Background ◽  
Plasmid Stability ◽  
Conjugation System ◽  
E Coli ◽  
Pantoea Stewartii ◽  
The Stability ◽  
Derivatives Of ◽  
Pilus Assembly

ABSTRACT Plasmid pSW100 is 1 of the 13 plasmids from Pantoea stewartii subsp. stewartii SW2 which has a replicon that resembles that of ColE1. This work uses a pSW100 derivative, pSW140K, to study how the pSW100 replicon is stably maintained in its hosts. Our results indicate that although pSW140K is stable in Escherichia coli HB101, the plasmid is rapidly lost in another E. coli strain, DH5α, indicating that the genetic background of an E. coli strain affects the stability of pSW140K. Mutagenesis of E. coli HB101 with EZ::TN <DHFR-1> revealed that mutations in traC, traF, traG, traN, and traV, which encode the components of the sex pilus assembly, reduce plasmid stability. Furthermore, this work identified that a 38-bp region located immediately upstream of the RNAII promoter is critical to the maintenance of plasmid stability in E. coli HB101. TraC binds to the region, and in addition, deleting the region destabilizes the plasmid. Furthermore, inserting this 38-bp fragment into a plasmid that contains the minimal replicon from pSW200 stabilizes the plasmid in E. coli HB101. Fluorescence in situ hybridization and immunofluorescence staining also revealed that derivatives of pSW100, pSW128A, and TraC are colocalized in cells, suggesting that pSW100 may use the sex pilus assembly as a partition apparatus to ensure the even distribution of the plasmid during cell division, which may thus maintain the plasmid's stability.

scholarly journals Interplay between IncF plasmids and topoisomerase mutations conferring quinolone resistance in the Escherichia coli ST131 clone: stability and resistance evolution

2021 ◽  
Author(s):  
Jose-Manuel Rodríguez-Martínez ◽  
Lorena Lopez-Cerero ◽  
Ana García-Duque ◽  
Jesus Rodriguez-Baño ◽  
Alvaro Pascual
Keyword(s):  
Escherichia Coli ◽  
Plasmid Stability ◽  
Luria Broth ◽  
Quinolone Resistance ◽  
Experimental Conditions ◽  
E Coli ◽  
The Stability ◽  
The Impact ◽  
Mutant Frequency

Abstract The Escherichia coli ST131 H30-Rx subclone vehicles CTX-M-15 plasmids and mutations in gyrA and parC conferring multidrug resistance successfully in the clinical setting. The aim of this study was (1) to investigate the relationship of specific topoisomerase mutations on the stability of IncF (CTX-M producing) plasmids using isogenic E. coli mutants and (2) to investigate the impact of the IncF-type plasmids present in the E. coli clone ST131 on the evolution of quinolone resistance. E. coli ATCC 25922 (background strain) and derived mutants encoding specific QRDR substitutions were used. Also, NGS-characterized IncFIA and IncFIB plasmids (encoding CTX-M genes) were included. Plasmid stability was evaluated by sequential dilutions into Luria broth medium without antibiotics for 7 days. Mutant frequency to ciprofloxacin was also evaluated. Moderate differences in the IncF plasmids stability were observed among E. coli ATCC 25922 and isogenic mutants. Under our experimental conditions, the fluctuation of bacteria harboring plasmids was less than 0.5-log(10) in all cases. In the mutant frequency tests, it was observed that the presence of these IncF plasmids increased this value significantly (10–1000-fold). Quinolone resistance substitutions in gyrA or parC genes, frequently found associated with E. coli clone ST131, do not modify the stability of ST131-associated IncFIA and IncFIB plasmids under in vitro conditions. IncF-type plasmids present in E. coli clone ST131 facilitate the selection of resistance to quinolones. These results are consistent with the clinical scenario in which the combination of resistance to quinolones and beta-lactams is highly frequent in the E. coli clone ST131.

scholarly journals A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit.

1994 ◽  
Vol 180 (6) ◽  
pp. 2147-2153 ◽  
Author(s):  
M Pizza ◽  
M R Fontana ◽  
M M Giuliani ◽  
M Domenighini ◽  
C Magagnoli ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cholera Toxin ◽  
Neutralizing Antibodies ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
E Coli ◽  
Heat Labile ◽  
A Subunit ◽  
Adp Ribosyltransferase ◽  
Derivatives Of

Escherichia coli enterotoxin (LT) and the homologous cholera toxin (CT) are A-B toxins that cause travelers' diarrhea and cholera, respectively. So far, experimental live and killed vaccines against these diseases have been developed using only the nontoxic B portion of these toxins. The enzymatically active A subunit has not been used because it is responsible for the toxicity and it is reported to induce a negligible titer of toxin neutralizing antibodies. We used site-directed mutagenesis to inactivate the ADP-ribosyltransferase activity of the A subunit and obtained nontoxic derivatives of LT that elicited a good titer of neutralizing antibodies recognizing the A subunit. These LT mutants and equivalent mutants of CT may be used to improve live and killed vaccines against cholera and enterotoxinogenic E. coli.

scholarly journals Enzymatic synthesis and phosphorolysis of 4(2)-thioxo- and 6(5)-azapyrimidine nucleosides by E. coli nucleoside phosphorylases

2016 ◽  
Vol 12 ◽  
pp. 2588-2601 ◽  
Author(s):  
Vladimir A Stepchenko ◽  
Anatoly I Miroshnikov ◽  
Frank Seela ◽  
Igor A Mikhailopulo
Keyword(s):  
Purine Nucleoside Phosphorylase ◽  
Reaction Conditions ◽  
E Coli ◽  
No Formation ◽  
Structural Peculiarities ◽  
Substrate Properties ◽  
Nucleoside Phosphorylases ◽  
Derivatives Of

The trans-2-deoxyribosylation of 4-thiouracil (4SUra) and 2-thiouracil (2SUra), as well as 6-azauracil, 6-azathymine and 6-aza-2-thiothymine was studied using dG and E. coli purine nucleoside phosphorylase (PNP) for the in situ generation of 2-deoxy-α-D-ribofuranose-1-phosphate (dRib-1P) followed by its coupling with the bases catalyzed by either E. coli thymidine (TP) or uridine (UP) phosphorylases. 4SUra revealed satisfactory substrate activity for UP and, unexpectedly, complete inertness for TP; no formation of 2’-deoxy-2-thiouridine (2SUd) was observed under analogous reaction conditions in the presence of UP and TP. On the contrary, 2SU, 2SUd, 4STd and 2STd are good substrates for both UP and TP; moreover, 2SU, 4STd and 2’-deoxy-5-azacytidine (Decitabine) are substrates for PNP and the phosphorolysis of the latter is reversible. Condensation of 2SUra and 5-azacytosine with dRib-1P (Ba salt) catalyzed by the accordant UP and PNP in Tris∙HCl buffer gave 2SUd and 2’-deoxy-5-azacytidine in 27% and 15% yields, respectively. 6-Azauracil and 6-azathymine showed good substrate properties for both TP and UP, whereas only TP recognizes 2-thio-6-azathymine as a substrate. 5-Phenyl and 5-tert-butyl derivatives of 6-azauracil and its 2-thioxo derivative were tested as substrates for UP and TP, and only 5-phenyl- and 5-tert-butyl-6-azauracils displayed very low substrate activity. The role of structural peculiarities and electronic properties in the substrate recognition by E. coli nucleoside phosphorylases is discussed.

scholarly journals In Vitro and In Vivo Assessment of the Potential of Escherichia coli Phages to Treat Infections and Survive Gastric Conditions

2021 ◽  
Vol 9 (9) ◽  
pp. 1869
Author(s):  
Joanna Kaczorowska ◽  
Eoghan Casey ◽  
Gabriele A. Lugli ◽  
Marco Ventura ◽  
David J. Clarke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Galleria Mellonella ◽  
Enterotoxigenic Escherichia Coli ◽  
E Coli ◽  
Composite Mixture ◽  
Ph Conditions ◽  
The Stability ◽  
Higher Sensitivity

Enterotoxigenic Escherichia coli (ETEC) and Shigella ssp. infections are associated with high rates of mortality, especially in infants in developing countries. Due to increasing levels of global antibiotic resistance exhibited by many pathogenic organisms, alternative strategies to combat such infections are urgently required. In this study, we evaluated the stability of five coliphages (four Myoviridae and one Siphoviridae phage) over a range of pH conditions and in simulated gastric conditions. The Myoviridae phages were stable across the range of pH 2 to 7, while the Siphoviridae phage, JK16, exhibited higher sensitivity to low pH. A composite mixture of these five phages was tested in vivo in a Galleria mellonella model. The obtained data clearly shows potential in treating E. coli infections prophylactically.

Interaction specificity between the chaperone and proteolytic components of the cyanobacterial Clp protease

2012 ◽  
Vol 446 (2) ◽  
pp. 311-320 ◽  
Author(s):  
Anders Tryggvesson ◽  
Frida M. Ståhlberg ◽  
Axel Mogk ◽  
Kornelius Zeth ◽  
Adrian K. Clarke
Keyword(s):  
Escherichia Coli ◽  
Active Sites ◽  
Terminal Sequence ◽  
Core Complex ◽  
Clp Protease ◽  
E Coli ◽  
Loop Region ◽  
N Terminus ◽  
Specific Association ◽  
The Stability

The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. In plant chloroplasts and cyanobacteria, the essential constitutive Clp protease consists of the Hsp100/ClpC chaperone partnering a proteolytic core of catalytic ClpP and noncatalytic ClpR subunits. In the present study, we have examined putative determinants conferring the highly specific association between ClpC and the ClpP3/R core from the model cyanobacterium Synechococcus elongatus. Two conserved sequences in the N-terminus of ClpR (tyrosine and proline motifs) and one in the N-terminus of ClpP3 (MPIG motif) were identified as being crucial for the ClpC–ClpP3/R association. These N-terminal domains also influence the stability of the ClpP3/R core complex itself. A unique C-terminal sequence was also found in plant and cyanobacterial ClpC orthologues just downstream of the P-loop region previously shown in Escherichia coli to be important for Hsp100 association to ClpP. This R motif in Synechococcus ClpC confers specificity for the ClpP3/R core and prevents association with E. coli ClpP; its removal from ClpC reverses this core specificity.

scholarly journals A Sequence That Affects the Copy Number and Stability of pSW200 and ColE1

2010 ◽  
Vol 192 (14) ◽  
pp. 3654-3660 ◽  
Author(s):  
Ying-Chung Wu ◽  
Shih-Tung Liu
Keyword(s):  
Copy Number ◽  
Plasmid Stability ◽  
Efficient Synthesis ◽  
Transcriptional Fusion ◽  
Similar Sequence ◽  
Pantoea Stewartii ◽  
The Stability

ABSTRACT Pantoea stewartii SW2 contains 13 plasmids. One of these plasmids, pSW200, has a replicon that resembles that of ColE1. This study demonstrates that pSW200 contains a 9-bp UP element, 5′-AAGATCTTC, which is located immediately upstream of the −35 box in the RNAII promoter. A transcriptional fusion study reveals that substituting this 9-bp sequence reduces the activity of the RNAII promoter by 78%. The same mutation also reduced the number of plasmid copies from 13 to 5, as well as the plasmid stability. When a similar sequence in a ColE1 derivative, pYCW301, is mutated, the copy number of the plasmid also declines from 34 to 16 per cell. Additionally, inserting this 9-bp sequence stabilizes an unstable pSW100 derivative, pSW142K, which also contains a replicon resembling that of ColE1, indicating the importance of this sequence in maintaining the stability of the plasmid. In conclusion, the 9-bp sequence upstream of the −35 box in the RNAII promoter is required for the efficient synthesis of RNAII and maintenance of the stability of the plasmids in the ColE1 family.

scholarly journals Overproduction of Penicillin-Binding Protein 2 and Its Inactive Variants Causes Morphological Changes and Lysis in Escherichia coli

2007 ◽  
Vol 189 (14) ◽  
pp. 4975-4983 ◽  
Author(s):  
Blaine A. Legaree ◽  
Calvin B. Adams ◽  
Anthony J. Clarke
Keyword(s):  
Escherichia Coli ◽  
Signal Sequence ◽  
Morphological Changes ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Prolonged Incubation ◽  
Lytic Transglycosylases ◽  
E Coli ◽  
Derivatives Of

ABSTRACT Penicillin-binding protein 2 (PBP 2) has long been known to be essential for rod-shaped morphology in gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. In the course of earlier studies with P. aeruginosa PBP 2, we observed that E. coli was sensitive to the overexpression of its gene, pbpA. In this study, we examined E. coli overproducing both P. aeruginosa and E. coli PBP 2. Growth of cells entered a stationary phase soon after induction of gene expression, and cells began to lyse upon prolonged incubation. Concomitant with the growth retardation, cells were observed to have changed morphologically from typical rods into enlarged spheres. Inactive derivatives of the PBP 2s were engineered, involving site-specific replacement of their catalytic Ser residues with Ala in their transpeptidase module. Overproduction of these inactive PBPs resulted in identical effects. Likewise, overproduction of PBP 2 derivatives possessing only their N-terminal non-penicillin-binding module (i.e., lacking their C-terminal transpeptidase module) produced similar effects. However, E. coli overproducing engineered derivatives of PBP 2 lacking their noncleavable, N-terminal signal sequence and membrane anchor were found to grow and divide at the same rate as control cells. The morphological effects and lysis were also eliminated entirely when overproduction of PBP 2 and variants was conducted with E. coli MHD79, a strain lacking six lytic transglycosylases. A possible interaction between the N-terminal domain of PBP 2 and lytic transglycosylases in vivo through the formation of multienzyme complexes is discussed.

scholarly journals High-Temperature Fluorescent In Situ Hybridization for Detecting Escherichia coli in Seawater Samples, Using rRNA-Targeted Oligonucleotide Probes and Flow Cytometry

2005 ◽  
Vol 71 (12) ◽  
pp. 8157-8164 ◽  
Author(s):  
Ying Zhong Tang ◽  
Karina Yew Hoong Gin ◽  
Tok Hoon Lim
Keyword(s):  
Escherichia Coli ◽  
Signal Intensity ◽  
Fluorescence Signal ◽  
Standard Protocol ◽  
Oligonucleotide Probes ◽  
E Coli ◽  
Hybridization Efficiency ◽  
Hybridization Time ◽  
Seawater Samples

ABSTRACT Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46°C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E.coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with a hybridization time of less than 30 min, with performance equivalent to or better than the standard protocol in terms of the fluorescence signal intensity and the FISH hybridization efficiency (i.e., the percentage of hybridized cells giving satisfactory fluorescence intensity): (i) one-step FISH (hybridization is conducted at 60 to 75°C for 30 min) and (ii) two-step FISH (pretreatment in a 90°C water bath for 5 min and a hybridizing step at 50 to 55°C for 15 to 20 min). We also found that satisfactory fluorescence signal intensity does not necessarily guarantee satisfactory hybridization efficiency and the tightness of the targeted population when analyzed with a flow cytometer. We subsequently successfully applied the optimized protocols to E. coli-spiked seawater samples, i.e., obtained flow cytometric signatures where the E. coli population was well separated from other particles carrying fluorescence from nonspecific binding to probes or from autofluorescence, and had a good recovery rate of the spiked E. coli cells (90%).

scholarly journals Cloning of the Human Cytomegalovirus (HCMV) Genome as an Infectious Bacterial Artificial Chromosome in Escherichia coli: a New Approach for Construction of HCMV Mutants

1999 ◽  
Vol 73 (10) ◽  
pp. 8320-8329 ◽  
Author(s):  
Eva-Maria Borst ◽  
Gabriele Hahn ◽  
Ulrich H. Koszinowski ◽  
Martin Messerle
Keyword(s):  
Escherichia Coli ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Artificial Chromosome ◽  
Bacterial Artificial Chromosomes ◽  
Reading Frame ◽  
E Coli ◽  
Artificial Chromosomes ◽  
Internal Repeat ◽  
The Stability

ABSTRACT We have recently introduced a novel procedure for the construction of herpesvirus mutants that is based on the cloning and mutagenesis of herpesvirus genomes as infectious bacterial artificial chromosomes (BACs) in Escherichia coli (M. Messerle, I. Crnković, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759–14763, 1997). Here we describe the application of this technique to the human cytomegalovirus (HCMV) strain AD169. Since it was not clear whether the terminal and internal repeat sequences of the HCMV genome would give rise to recombination, the stability of the cloned HCMV genome was examined during propagation inE. coli, during mutagenesis, and after transfection in permissive fibroblasts. Interestingly, the HCMV BACs were frozen in defined conformations in E. coli. The transfection of the HCMV BACs into human fibroblasts resulted in the reconstitution of infectious virus and isomerization of the reconstituted genomes. The power of the BAC mutagenesis procedure was exemplarily demonstrated by the disruption of the gpUL37 open reading frame. The transfection of the mutated BAC led to plaque formation, indicating that the gpUL37 gene product is dispensable for growth of HCMV in fibroblasts. The new procedure will considerably speed up the construction of HCMV mutants and facilitate genetic analysis of HCMV functions.

scholarly journals Periplasmic Peptidyl Prolyl cis-trans Isomerases Are Not Essential for Viability, but SurA Is Required for Pilus Biogenesis in Escherichia coli

2005 ◽  
Vol 187 (22) ◽  
pp. 7680-7686 ◽  
Author(s):  
Sheryl S. Justice ◽  
David A. Hunstad ◽  
Jill Reiss Harper ◽  
Amy R. Duguay ◽  
Jerome S. Pinkner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane Proteins ◽  
E Coli ◽  
Outer Membranes ◽  
Type 1 Pili ◽  
Pilus Biogenesis ◽  
Quadruple Mutant ◽  
Increased Susceptibility ◽  
Pilus Assembly

ABSTRACT In Escherichia coli, FkpA, PpiA, PpiD, and SurA are the four known periplasmic cis-trans prolyl isomerases. These isomerases facilitate proper protein folding by increasing the rate of transition of proline residues between the cis and trans states. Genetic inactivation of all four periplasmic isomerases resulted in a viable strain that exhibited a decreased growth rate and increased susceptibility to certain antibiotics. Levels of the outer membrane proteins LamB and OmpA in the quadruple mutant were indistinguishable from those in the surA single mutant. In addition, expression of P and type 1 pili (adhesive organelles produced by uropathogenic strains of E. coli and assembled by the chaperone/usher pathway) were severely diminished in the absence of the four periplasmic isomerases. Maturation of the usher was significantly impaired in the outer membranes of strains devoid of all four periplasmic isomerases, resulting in a defect in pilus assembly. Moreover, this defect in pilus assembly and usher stability could be attributed to the absence of SurA. The data presented here suggest that the four periplasmic isomerases are not essential for growth under laboratory conditions but may have significant roles in survival in environmental and pathogenic niches, as indicated by the effect on pilus production.

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