scholarly journals Functional Analysis of the Acinetobacter baumannii XerC and XerD Site-Specific Recombinases: Potential Role in Dissemination of Resistance Genes

Antibiotics ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 405
Author(s):  
David L. Lin ◽  
German M. Traglia ◽  
Rachel Baker ◽  
David J. Sherratt ◽  
Maria Soledad Ramirez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Functional Analysis ◽  
Acinetobacter Baumannii ◽  
Potential Role ◽  
Covalent Attachment ◽  
Recombination Reaction ◽  
Site Specific ◽  
Site Specific Recombination ◽  
E Coli ◽  
Resistant Genes

Modules composed of a resistance gene flanked by Xer site-specific recombination sites, the vast majority of which were found in Acinetobacter baumannii, are thought to behave as elements that facilitate horizontal dissemination. The A. baumannii xerC and xerD genes were cloned, and the recombinant clones used to complement the cognate Escherichia coli mutants. The complemented strains supported the resolution of plasmid dimers, and, as is the case with E. coli and Klebsiella pneumoniae plasmids, the activity was enhanced when the cells were grown in a low osmolarity growth medium. Binding experiments showed that the partially purified A. baumannii XerC and XerD proteins (XerCAb and XerDAb) bound synthetic Xer site-specific recombination sites, some of them with a nucleotide sequence deduced from existing A. baumannii plasmids. Incubation with suicide substrates resulted in the covalent attachment of DNA to a recombinase, probably XerCAb, indicating that the first step in the recombination reaction took place. The results described show that XerCAb and XerDAb are functional proteins and support the hypothesis that they participate in horizontal dissemination of resistant genes among bacteria.

scholarly journals Functional Analysis of the Acinetobacter baumannii XerC and XerD Site-Specific Recombinases: Potential Role in Dissemination of Resistance Genes

2020 ◽  
Author(s):  
David L. Lin ◽  
German M. Traglia ◽  
Rachel Baker ◽  
David J. Sherratt ◽  
Maria S. Ramirez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Functional Analysis ◽  
Acinetobacter Baumannii ◽  
Potential Role ◽  
Covalent Attachment ◽  
Recombination Reaction ◽  
Site Specific ◽  
Site Specific Recombination ◽  
E Coli ◽  
Resistant Genes

Modules composed of a resistance gene flanked by Xer site-specific recombination sites, the vast majority of which were found in Acinetobacter baumannii, are thought to behave as elements that facilitate horizontal dissemination. The xerCAb and xerDAb genes were cloned, and the recombinant clones used to complement the cognate Escherichia coli mutants. The complemented strains supported resolution of plasmid dimers, and, as is the case with E. coli and Klebsiella pneumoniae plasmids, the activity was enhanced when cells were growing in low osmolarity growth medium. Binding experiments showed that partially purified A. baumannii XerC and XerD proteins (XerCAb and XerDAb) bound synthetic Xer site-specific recombination sites, some of them with a nucleotide sequence deduced from existing A. baumannii plasmids. Incubation with suicide substrates resulted in covalent attachment of DNA to a recombinase, probably XerCAb, indicating that the first step in the recombination reaction took place. The results described show that XerCAb and XerDAb are functional proteins that actively participate in horizontal dissemination of resistant genes among bacteria.

scholarly journals Osmoregulation of Dimer Resolution at the Plasmid pJHCMW1 mwr Locus by Escherichia coli XerCD Recombination

2002 ◽  
Vol 184 (6) ◽  
pp. 1607-1616 ◽  
Author(s):  
Huong Pham ◽  
Ken J. Dery ◽  
David J. Sherratt ◽  
Marcelo E. Tolmasky
Keyword(s):  
Escherichia Coli ◽  
Central Region ◽  
High Salt ◽  
Recombination Reaction ◽  
Recombination Site ◽  
Site Specific ◽  
Site Specific Recombination ◽  
E Coli ◽  
Low Salt ◽  
Ionic Effects

ABSTRACT Xer-mediated dimer resolution at the mwr site of plasmid pJHCMW1 is osmoregulated in Escherichia coli. Whereas under low-salt conditions, the site-specific recombination reaction is efficient, under high-salt conditions, it proceeds inefficiently. Regulation of dimer resolution is independent of H-NS and is mediated by changes in osmolarity rather than ionic effects. The low level of recombination at high salt concentrations can be overcome by high levels of PepA or by mutating the ARG box to a sequence closer to the E. coli ARG box consensus. The central region of the mwr core recombination site plays a role in regulation of site-specific recombination by the osmotic pressure of the medium.

scholarly journals The Escherichia coli Fis Protein Stimulates Bacteriophage λ Integrative Recombination In Vitro

2003 ◽  
Vol 185 (10) ◽  
pp. 3076-3080 ◽  
Author(s):  
Dominic Esposito ◽  
Gary F. Gerard
Keyword(s):  
Escherichia Coli ◽  
Host Cell ◽  
Essential Role ◽  
Bacteriophage Λ ◽  
Site Specific ◽  
Site Specific Recombination ◽  
E Coli ◽  
In Vivo Recombination

ABSTRACT The Escherichia coli nucleoid-associated protein Fis was previously shown to be involved in bacteriophage lambda site-specific recombination in vivo, enhancing the levels of both integrative recombination and excisive recombination. While purified Fis protein was shown to stimulate in vitro excision, Fis appeared to have no effect on in vitro integration reactions even though a 15-fold drop in lysogenization frequency had previously been observed in fis mutants. We demonstrate here that E. coli Fis protein does stimulate integrative lambda recombination in vitro but only under specific conditions which likely mimic natural in vivo recombination more closely than the standard conditions used in vitro. In the presence of suboptimal concentrations of Int protein, Fis stimulates the rate of integrative recombination significantly. In addition, Fis enhances the recombination of substrates with nonstandard topologies which may be more relevant to the process of in vivo phage lambda recombination. These data support the hypothesis that Fis may play an essential role in lambda recombination in the host cell.

scholarly journals Site-Specific Recombination in the Cyanobacterium Anabaena sp. Strain PCC 7120 Catalyzed by the Integrase of Coliphage HK022

2009 ◽  
Vol 191 (13) ◽  
pp. 4458-4464 ◽  
Author(s):  
Olga Melnikov ◽  
Arieh Zaritsky ◽  
Aliza Zarka ◽  
Sammy Boussiba ◽  
Natalia Malchin ◽  
...  
Keyword(s):  
Integration Host Factor ◽  
Specific Gene ◽  
Recombination Reaction ◽  
Site Specific ◽  
Site Specific Recombination ◽  
E Coli ◽  
Site Specific Integration ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120

ABSTRACT The integrase (Int) of the λ-like coliphage HK022 catalyzes the site-specific integration and excision of the phage DNA into and from the chromosome of its host, Escherichia coli. Int recognizes two different pairs of recombining sites attP × attB and attL × attR for integration and excision, respectively. This system was adapted to the cyanobacterium Anabaena sp. strain PCC 7120 as a potential tool for site-specific gene manipulations in the cyanobacterium. Two plasmids were consecutively cointroduced by conjugation into Anabaena cells, one plasmid that expresses HK022 Int recombinase and the other plasmid that carries the excision substrate PglnA -attL-T1/T2-attR-lacZ, where T1/T2 are the strong transcription terminators of rrnB, to prevent expression of the lacZ reporter under the constitutive promoter PglnA . The Int-catalyzed site-specific recombination reaction was monitored by the expression of lacZ emanating as a result of T1/T2 excision. Int catalyzed the site-specific excision reaction in Anabaena cells when its substrate was located either on the plasmid or on the chromosome with no need to supply an accessory protein, such as integration host factor and excisionase (Xis), which are indispensable for this reaction in its host, E. coli.

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 Mechanistic insights into synergy between nalidixic acid and tetracycline against clinical isolates of Acinetobacter baumannii and Escherichia coli

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Amit Gaurav ◽  
Varsha Gupta ◽  
Sandeep K. Shrivastava ◽  
Ranjana Pathania
Keyword(s):  
Escherichia Coli ◽  
Acinetobacter Baumannii ◽  
Nalidixic Acid ◽  
Bacterial Infections ◽  
Clinical Isolates ◽  
Hospital Environment ◽  
Infection Model ◽  
Drug Resistant ◽  
E Coli

AbstractThe increasing prevalence of antimicrobial resistance has become a global health problem. Acinetobacter baumannii is an important nosocomial pathogen due to its capacity to persist in the hospital environment. It has a high mortality rate and few treatment options. Antibiotic combinations can help to fight multi-drug resistant (MDR) bacterial infections, but they are rarely used in the clinics and mostly unexplored. The interaction between bacteriostatic and bactericidal antibiotics are mostly reported as antagonism based on the results obtained in the susceptible model laboratory strain Escherichia coli. However, in the present study, we report a synergistic interaction between nalidixic acid and tetracycline against clinical multi-drug resistant A. baumannii and E. coli. Here we provide mechanistic insight into this dichotomy. The synergistic combination was studied by checkerboard assay and time-kill curve analysis. We also elucidate the mechanism behind this synergy using several techniques such as fluorescence spectroscopy, flow cytometry, fluorescence microscopy, morphometric analysis, and real-time polymerase chain reaction. Nalidixic acid and tetracycline combination displayed synergy against most of the MDR clinical isolates of A. baumannii and E. coli but not against susceptible isolates. Finally, we demonstrate that this combination is also effective in vivo in an A. baumannii/Caenorhabditis elegans infection model (p < 0.001)

Two related recombinases are required for site-specific recombination at dif and cer in E. coli K12

Cell ◽  
1993 ◽  
Vol 75 (2) ◽  
pp. 351-361 ◽  
Author(s):  
Garry Blakely ◽  
Gerhard May ◽  
Richard McCulloch ◽  
Lidia K. Arciszewska ◽  
Mary Burke ◽  
...  
Keyword(s):  
Site Specific ◽  
Site Specific Recombination ◽  
E Coli

An Escherichia coli system for assay of Flp site-specific recombination on substrate plasmids

Gene ◽  
1996 ◽  
Vol 180 (1-2) ◽  
pp. 225-227 ◽  
Author(s):  
Michael R. Snaith ◽  
Nigel J. Kilby ◽  
James A.H. Murray
Keyword(s):  
Escherichia Coli ◽  
Site Specific ◽  
Site Specific Recombination

scholarly journals The Site-Specific Recombination System of the Escherichia coli Bacteriophage Φ24B

2020 ◽  
Vol 11 ◽  
Author(s):  
Mohammed Radhi Mohaisen ◽  
Alan John McCarthy ◽  
Evelien M. Adriaenssens ◽  
Heather Elizabeth Allison
Keyword(s):  
Escherichia Coli ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Recombination System ◽  
Site Specific Recombination System

scholarly journals Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement

Biomolecules ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 255 ◽  
Author(s):  
Sviatlana Smolskaya ◽  
Yaroslav Andreev
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Trna Synthetase ◽  
Nonsense Suppression ◽  
Expression Vectors ◽  
Site Specific ◽  
Suppressor Trna ◽  
E Coli ◽  
Orthogonal Pair

More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E. coli with the emphasis on the improved expression vectors encoding for an orthogonal aa-RA/tRNA pair, enhancement of aa-RS and suppressor tRNA efficiency, the evolution of orthogonal EF-Tu and attempts to reduce the effect of RF1.

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