scholarly journals The Large Resolvase TndX Is Required and Sufficient for Integration and Excision of Derivatives of the Novel Conjugative Transposon Tn5397

2000 ◽  
Vol 182 (23) ◽  
pp. 6577-6583 ◽  
Author(s):  
Hongmei Wang ◽  
Peter Mullany
Keyword(s):  
Hot Spots ◽  
Activity Analysis ◽  
The Novel ◽  
Conjugation System ◽  
Conjugative Transposon ◽  
Site Specific ◽  
E Coli ◽  
Derivatives Of ◽  
Conserved Amino Acids

ABSTRACT Tn5397 is a novel conjugative transposon, originally isolated from Clostridium difficile. This element can transfer between C. difficile strains and to and fromBacillus subtilis. It encodes a conjugation system that is very similar to that of Tn916. However, insertion and excision of Tn5397 appears to be dependent on the product of the element encoded gene tndX, a member of the large resolvase family of site-specific recombinases. To test the role oftndX, the gene was cloned and the protein was expressed inEscherichia coli. The ability of TndX to catalyze the insertion and excision of derivatives (minitransposons) of Tn5397 representing the putative circular and integrated forms, respectively, was investigated. TndX was required for both insertion and excision. Mutagenesis studies showed that some of the highly conserved amino acids at the N-terminal resolvase domain and the C-terminal nonconserved region of TndX are essential for activity. Analysis of the target site choices showed that the cloned Tn5397 targets from C. difficile and B. subtilis were still hot spots for the minitransposon insertion inE. 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 Pathoadaptive Mutations That Enhance Virulence: Genetic Organization of the cadA Regions ofShigella spp

2001 ◽  
Vol 69 (12) ◽  
pp. 7471-7480 ◽  
Author(s):  
William A. Day ◽  
Reinaldo E. Fernández ◽  
Anthony T. Maurelli
Keyword(s):  
The Novel ◽  
Gene Encoding ◽  
E Coli ◽  
Novel Gene ◽  
Genetic Linkages ◽  
Important Pathway ◽  
K 12 ◽  
Pathoadaptive Mutations ◽  
Host Tissues

ABSTRACT Pathoadaptive mutations improve the fitness of pathogenic species by modification of traits that interfere with factors (virulence and ancestral) required for survival in host tissues. A demonstrated pathoadaptive mutation is the loss of lysine decarboxylase (LDC) expression in Shigella species that have evolved from LDC-expressing Escherichia coli. Previous studies demonstrated that the product of LDC activity, cadaverine, blocks the action of Shigella enterotoxins and that the gene encoding LDC, cadA, was abolished by large chromosomal deletions in each Shigella species. To better understand the nature and evolution of these pathoadaptive mutations, remnants of thecad region were sequenced from the fourShigella species. These analyses reveal novel gene arrangements in this region of the pathogens' chromosomes. Insertion sequences, a phage genome, and/or loci from different positions on the ancestral E. coli chromosome displaced the cadAlocus to form distinct genetic linkages that are unique to eachShigella species. Hybridization studies, using an E. coli K-12 microarray, indicated that the genes displaced to form the novel linkages still remain in the Shigella genomes. None of these novel gene arrangements were observed in representatives of all E. coli phylogenies. Collectively, these observations indicate that inactivation of the cadAantivirulence gene occurred independently in each Shigellaspecies. The convergent evolution of these pathoadaptive mutations demonstrates that, following evolution from commensal E. coli, strong pressures in host tissues selectedShigella clones with increased fitness and virulence through the loss of an ancestral trait (LDC). These observations strongly support the role of pathoadaptive mutation as an important pathway in the evolution of pathogenic organisms.

scholarly journals Instability of Pathogenicity Islands in Uropathogenic Escherichia coli 536

2004 ◽  
Vol 186 (10) ◽  
pp. 3086-3096 ◽  
Author(s):  
Barbara Middendorf ◽  
Bianca Hochhut ◽  
Kristina Leipold ◽  
Ulrich Dobrindt ◽  
Gabriele Blum-Oehler ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Direct Repeat ◽  
Pathogenicity Islands ◽  
Site Specific ◽  
Site Specific Recombination ◽  
E Coli ◽  
Specific Pcr ◽  
Specific Pcr Assay ◽  
Derivatives Of ◽  
Specific Sequences

ABSTRACT The uropathogenic Escherichia coli strain 536 carries at least five genetic elements on its chromosome that meet all criteria characteristic of pathogenicity islands (PAIs). One main feature of these distinct DNA regions is their instability. We applied the so-called island-probing approach and individually labeled all five PAIs of E. coli 536 with the counterselectable marker sacB to evaluate the frequency of PAI-negative colonies under the influence of different environmental conditions. Furthermore, we investigated the boundaries of these PAIs. According to our experiments, PAI II536 and PAI III536 were the most unstable islands followed by PAI I536 and PAI V536, whereas PAI IV536 was stable. In addition, we found that deletion of PAI II536 and PAI III536 was induced by several environmental stimuli. Whereas excision of PAI I536, PAI II536, and PAI V536 was based on site-specific recombination between short direct repeat sequences at their boundaries, PAI III536 was deleted either by site-specific recombination or by homologous recombination between two IS100-specific sequences. In all cases, deletion is thought to lead to the formation of nonreplicative circular intermediates. Such extrachromosomal derivatives of PAI II536 and PAI III536 were detected by a specific PCR assay. Our data indicate that the genome content of uropathogenic E. coli can be modulated by deletion of PAIs.

scholarly journals Ciprofloxacin facilitates the transfer of XDR plasmids from commensal E. coli into epidemic fluoroquinolone-resistant Shigella sonnei

2019 ◽  
Author(s):  
Pham Thanh Duy ◽  
To Nguyen Thi Nguyen ◽  
Duong Vu Thuy ◽  
Hao Chung The ◽  
Felicity Alcock ◽  
...  
Keyword(s):  
Diarrheal Disease ◽  
Treatment Guidelines ◽  
Healthy Children ◽  
The Novel ◽  
Shotgun Metagenomics ◽  
E Coli ◽  
High Prevalence ◽  
New Locations ◽  
Third Generation Cephalosporins

AbstractThe global dissemination of a ciprofloxacin-resistant (cipR) S. sonnei clone outlines the mobility of this important agent of diarrheal disease, and threatens the utility of ciprofloxacin as a first-line antimicrobial for shigellosis. Here, we aimed to track the emergence of cipR S. sonnei in Vietnam to understand how novel antimicrobial resistant (AMR) Shigella clones become established in new locations. From 2014 to 2016, we isolated and genome sequenced 79 S. sonnei from children hospitalized with dysenteric diarrhea in southern Vietnam. The novel cipR S. sonnei clone displaced the resident ciprofloxacin-susceptible lineage while acquiring resistance against third-generation cephalosporins, macrolides, and aminoglycosides. This process was not the result of a single clonal expansion, as we identified at least thirteen independent acquisitions of ESBL-encoding plasmids. The frequency and diversity of the variable AMR repertoire in an expanding clonal background of S. sonnei is unprecedented and we speculated that it was facilitated by horizontal gene transfer from commensal organisms in the human gut. Consequently, we characterized non-Shigella Enterobacteriaceae from Shigella-infected and healthy children by shotgun metagenomics. We identified a wide array of AMR genes and plasmids in the commensal Enterobacteriaceae, including an E. coli isolated from a Shigella-infected child with an identical ESBL plasmid to that characterized in the infecting S. sonnei. We confirmed that these AMR plasmids could be exchanged between commensal E. coli and S. sonnei and found that supplementation of ciprofloxacin into the conjugation media significantly increased the conjugation frequency of IncI/blaCTX-M-15, IncB/O/blaCTX-M-27 and IncF/blaCTX-M-27 plasmids. In a setting with high antimicrobial use and a high prevalence of AMR commensals, cipR S. sonnei may be propelled towards pan-resistance by adherence to outdated international treatment guidelines. Our work highlights the role of the gut microbiota in transferring resistance plasmids into enteric pathogens and provides essential data to restrict the use of ciprofloxacin globally.

scholarly journals Identification and Analysis of “Extended −10” Promoters from Mycobacteria

1998 ◽  
Vol 180 (9) ◽  
pp. 2568-2573 ◽  
Author(s):  
Murali D. Bashyam ◽  
Anil K. Tyagi
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Sigma Factor ◽  
Mycobacterium Smegmatis ◽  
Important Determinant ◽  
Comparative Assessment ◽  
Binding Domain ◽  
Site Specific ◽  
E Coli

ABSTRACT Earlier studies from our laboratory on randomly isolated transcriptional signals of mycobacteria had revealed that the −10 region of mycobacterial promoters and the corresponding binding domain in the major sigma factor are highly similar to their Escherichia coli counterparts. In contrast, the sequences in −35 regions of mycobacterial promoters and the corresponding binding domain in the major sigma factor are vastly different from their E. colicounterparts (M. D. Bashyam, D. Kaushal, S. K. Dasgupta, and A. K. Tyagi, J. Bacteriol. 178:4847–4853, 1996). We have now analyzed the role of the TGN motif present immediately upstream of the −10 region of mycobacterial promoters. Sequence analysis and site-specific mutagenesis of a Mycobacterium tuberculosispromoter and a Mycobacterium smegmatis promoter reveal that the TGN motif is an important determinant of transcriptional strength in mycobacteria. We show that mutation in the TGN motif can drastically reduce the transcriptional strength of a mycobacterial promoter. The influence of the TGN motif on transcriptional strength is also modulated by the sequences in the −35 region. Comparative assessment of these extended −10 promoters in mycobacteria and E. coli suggests that functioning of the TGN motif in promoters of these two species is similar.

scholarly journals Construction and Characterization of an Isogenicslt-ii Deletion Mutant of EnterohemorrhagicEscherichia coli

1998 ◽  
Vol 66 (5) ◽  
pp. 2337-2341 ◽  
Author(s):  
Florian Gunzer ◽  
Ursula Bohn ◽  
Sibylle Fuchs ◽  
Inge Mühldorfer ◽  
Jörg Hacker ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Deletion Mutant ◽  
Protein Synthesis Inhibitors ◽  
Course Of Disease ◽  
E Coli ◽  
Suicide Vector ◽  
Derivatives Of ◽  
Negative Mutant

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) produces Shiga-like toxins (SLT), potent protein synthesis inhibitors. To further dissect the role of SLT-II in the course of disease, we have constructed E. coli TUV86-2, an isogenic SLT-II-negative mutant of EHEC strain 86-24. The slt-ii gene was inactivated by suicide vector mutagenesis. We also isolated derivatives of strain 86-24 that were cured of the phage carrying the toxin genes.

scholarly journals Sister Chromatid Exchange Frequencies inEscherichia coli Analyzed by Recombination at thedif Resolvase Site

1998 ◽  
Vol 180 (23) ◽  
pp. 6269-6275 ◽  
Author(s):  
Walter W. Steiner ◽  
Peter L. Kuempel
Keyword(s):  
Sister Chromatid ◽  
Sister Chromatid Exchange ◽  
Oxidative Dna Damage ◽  
Inescherichia Coli ◽  
Wild Type ◽  
Site Specific ◽  
Factors Affecting ◽  
E Coli ◽  
Chromosomal Recombination

ABSTRACT Sister chromatid exchange (SCE) in Escherichia coliresults in the formation of circular dimer chromosomes, which are converted back to monomers by a compensating exchange at thedif resolvase site. Recombination at dif is site specific and can be monitored by utilizing a density label assay that we recently described. To characterize factors affecting SCE frequency, we analyzed dimer resolution at the dif site in a variety of genetic backgrounds and conditions. Recombination atdif was increased by known hyperrecombinogenic mutations such as polA, dut, and uvrD. It was also increased by a fur mutation, which increased oxidative DNA damage. Recombination at dif was eliminated by arecA mutation, reflecting the role of RecA in SCE and virtually all homologous recombination in E. coli. Interestingly, recombination at dif was reduced to approximately half of the wild-type levels by single mutations in either recB or recF, and it was virtually eliminated when both mutations were present. This result demonstrates the importance of both RecBCD and RecF to chromosomal recombination events in wild-type cells.

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 1727. Sustained Antimicrobial Activity of a Novel Disinfectant Against Healthcare Pathogens

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S55-S55 ◽  
Author(s):  
William Rutala ◽  
Maria Gergen ◽  
Emily Sickbert-Bennett ◽  
Deverick J Anderson ◽  
David Weber
Keyword(s):  
Hydrogen Peroxide ◽  
Antimicrobial Activity ◽  
Stainless Steel ◽  
The Novel ◽  
Log10 Reduction ◽  
Final Time ◽  
E Coli ◽  
Environmental Surfaces ◽  
The Mean

Abstract Background Environmental contamination plays an important role in the transmission of MRSA, VRE, and C. difficile. Suboptimal compliance with hand hygiene or inappropriate glove use can result in indirect transfer of these pathogens to patients. This study evaluates a novel disinfectant that claims to kill microbes on surfaces for ≥24 hours. Methods We investigated the persistent antimicrobial activity of a novel disinfectant using an EPA protocol for sustained disinfecting activity. In brief, surfaces are inoculated, treated with the novel disinfectant, allowed to dry, and then abraded using a standardized abrasion machine under multiple alternating wet and dry wipe conditions (N = 12) interspersed with 6 re-inoculations. After 24 hours, the surface was re-inoculated a final time and ability of the disinfectant to kill ≥99.9% of 9 test microbes within 5 minutes was measured on 3 test surfaces (glass, formica, and stainless steel). Results The novel disinfectant demonstrated a 3–5 log10 reduction in 5 minutes when testing S. aureus, VRE, C. auris, CRE E. coli and antibiotic-sensitive strains of E. coli, and Enterobacter sp. (table). The disinfectant demonstrated lower killing for CRE isolates of Enterobacter sp. and K. pneumoniae, and for antibiotic-sensitive K. pneumoniae (~2 log10 reduction in 5 minutes). When the novel disinfectant was compared with 3 other commonly used disinfectants using the same methodology with S. aureus, the mean log10 reductions were: 4.4 (novel disinfectant); 0.9 (quat-alcohol); 0.2 (improved hydrogen peroxide); and 0.1 (chlorine). Conclusion Persistent disinfectants may reduce or eliminate the problem of recontamination and minimize the role of environmental surfaces in transmission of healthcare pathogens. Disclosures W. Rutala, PDI: Consultant and Speaker’s Bureau, Consulting fee and Speaker honorarium. D. Weber, PDI: Consultant, Consulting fee

scholarly journals Insights into Enzymic Catalysis from Studies on Dihydrofolate Reductases

Pteridines ◽  
1989 ◽  
Vol 1 (1) ◽  
pp. 37-43 ◽  
Author(s):  
Stephen J. Benkovic ◽  
Joseph A. Adams ◽  
Carol A. Fierke ◽  
Adel M. Naylor
Keyword(s):  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Structural Similarity ◽  
E Coli ◽  
Energy Profiles ◽  
Dihydrofolate Reductases ◽  
Cellular Dna ◽  
High Degree ◽  
Conserved Amino Acids

Summary The role of DHFR in the maintenance of cellular DNA has sparked wide interest in the structure and dynamics of this enzyme. Kinetic studies of specific amino acid replacements on the enzyme isolated from E. coli has proved useful in the detailing of hydrophobic and ionic interactions both proximal and distal to the site of chemical transformation (e. g. Phe-31, Leu-54 and Arg-44). Despite the low sequence homology shared by the E. coli and L. easei enzymes, the free energy profiles are surprisingly comparable. This probably is the result of the high degree of structural similarity of the active site surfaces, but the deleterious effects of subtle replacements (e. g. Leu-54-Ile) at strictly conserved amino acids underscore the latters unique role in attaining the required catalytic efficiency for the enzyme.

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