Cooperative binding of Tn3 resolvase monomers to a functionally asymmetric binding site

Blake, David G.; Boocock, Martin R.; Sherratt, David J.; Stark, W. Marshall

doi:10.1016/s0960-9822(95)00208-9

Cited by 28 publications

(32 citation statements)

References 36 publications

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“…In plasmid pAs(V)21, the gene encoding the ␤-lactamase BL is located between two mobile elements characterized as a TnpA transposase and a resolvase, enzymes involved in the transposition and replication of the Tn3 transposon group (28). Also on pAs(V)21, the gene for the unspecific macrolide transporter macA is located immediately downstream from a sequence encoding the subunit B of the transposase orfAB.…”

Section: Resultsmentioning

confidence: 99%

Natural Hot Spots for Gain of Multiple Resistances: Arsenic and Antibiotic Resistances in Heterotrophic, Aerobic Bacteria from Marine Hydrothermal Vent Fields

Farias

Santo²,

Branco³

et al. 2015

Appl Environ Microbiol

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dMicroorganisms are responsible for multiple antibiotic resistances that have been associated with resistance/tolerance to heavy metals, with consequences to public health. Many genes conferring these resistances are located on mobile genetic elements, easily exchanged among phylogenetically distant bacteria. The objective of the present work was to isolate arsenic-, antimonite-, and antibiotic-resistant strains and to determine the existence of plasmids harboring antibiotic/arsenic/antimonite resistance traits in phenotypically resistant strains, in a nonanthropogenically impacted environment. The hydrothermal Lucky Strike field in the Azores archipelago (North Atlantic, between 11°N and 38°N), at the Mid-Atlantic Ridge, protected under the OSPAR Convention, was sampled as a metal-rich pristine environment. A total of 35 strains from 8 different species were isolated in the presence of arsenate, arsenite, and antimonite. ACR3 and arsB genes were amplified from the sediment's total DNA, and 4 isolates also carried ACR3 genes. Phenotypic multiple resistances were found in all strains, and 7 strains had recoverable plasmids. Purified plasmids were sequenced by Illumina and assembled by EDENA V3, and contig annotation was performed using the "Rapid Annotation using the Subsystems Technology" server. Determinants of resistance to copper, zinc, cadmium, cobalt, and chromium as well as to the antibiotics ␤-lactams and fluoroquinolones were found in the 3 sequenced plasmids. Genes coding for heavy metal resistance and antibiotic resistance in the same mobile element were found, suggesting the possibility of horizontal gene transfer and distribution of theses resistances in the bacterial population.T he ocean floor near the Azores is constituted in part by the Mid-Atlantic Ridge and a series of extensive hydrothermal vents. The largest hydrothermal vent field known at this time is located at 37°18.5=N, 32°16.5=W, averages 1,700 m in depth, and is named "Lucky Strike" (1). Here, 21 active hydrothermal vents are connected to the thermal anomalies in the subsea floor at the Mid-Atlantic Ridge that are spread over 150 km 2 . The field is primarily made of basalt but also spikes interest due to rich deposits of ores. Temperatures of the field vary from 170°to 324°C (2), diminishing with the distance from the vent outlet due to water circularization with the surrounding cold temperatures characteristic of these depths (around 4°C), which therefore harbor a distinct microbial community. Furthermore, hydrothermal activity at Lucky Strike seems to have been episodic over a long time (2).The nature of the bedrock and the geological events at Lucky Strike vent sites result in smaller concentrations of Rn and heavy metals (Cd, Hg, Cu, Pb, Zn, Fe, and Ag) and less acidic fluids, making it one of the least extreme environments, in a biological perspective, of the Mid-Atlantic Ridge compared to neighboring vent sites (3).Antibiotics in the environment may result from adaptive phenotypic and genotypic responses of the microbiota. Recen...

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Section: Resultsmentioning

confidence: 99%

Natural Hot Spots for Gain of Multiple Resistances: Arsenic and Antibiotic Resistances in Heterotrophic, Aerobic Bacteria from Marine Hydrothermal Vent Fields

Farias

Santo²,

Branco³

et al. 2015

Appl Environ Microbiol

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“…2), which has been described (15). The substrates used for in vitro Z-site recombination assays were constructed similarly to pOG3, from the cloning vector pMTL23 (17). Full details of the plasmid construction and sequences are available on request.…”

Section: Methodsmentioning

confidence: 99%

Chimeric recombinases with designed DNA sequence recognition

Akopian

Boocock

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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107

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Site-specific recombination typically occurs only between DNA sequences that have co-evolved with a natural recombinase enzyme to optimize sequence recognition, catalytic efficiency, and regulation. Here, we show that the sequence recognition and the catalysis functions of a recombinase can be specified by unrelated protein domains. We describe chimeric recombinases with a catalytic domain from an activated multiple mutant of the bacterial enzyme Tn3 resolvase, fused to a DNA recognition domain from the mouse transcription factor Zif268. These proteins catalyze efficient recombination specifically at synthetic target sites recognized by two Zif268 domains. Our results demonstrate the functional autonomy of the resolvase catalytic domain and open the way to creating ''custom-built'' recombinases that act at chosen natural target sequences.

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“…The res sequence is altered from the natural sequence of Tn3 at three base-pairs in the DNA between binding sites I and II, creating a unique EcoRI site. This modi®ed res behaves normally in resolution assays (Blake et al, 1995). pMA21 was digested with EcoRI, the ends were ®lled using Klenow fragment DNA polymerase, and the DNA was further digested with HindIII.…”

Section: Methodsmentioning

confidence: 99%

“…The intensities of bands on ethidium-stained agarose gels were estimated by scanning photographic negatives with a Joyce-Loebl microdensitometer. Gel binding assays and autoradiography (Blake et al, 1995) and synapsis assays (Watson et al, 1996) were as described.…”

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confidence: 99%

Tn3 resolvase catalyses multiple recombination events without intermediate rejoining of DNA ends 1 1 Edited by J. Karn

McIlwraith

Boocock

Stark

1997

Journal of Molecular Biology

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Cooperative binding of Tn3 resolvase monomers to a functionally asymmetric binding site

Abstract: Deviations from perfect inverted-repeat symmetry in a resolvase-binding site lead to ordered binding of subunits, structural asymmetry of resolvase-DNA complexes, and asymmetric function.

Cited by 28 publications

References 36 publications

Natural Hot Spots for Gain of Multiple Resistances: Arsenic and Antibiotic Resistances in Heterotrophic, Aerobic Bacteria from Marine Hydrothermal Vent Fields

Natural Hot Spots for Gain of Multiple Resistances: Arsenic and Antibiotic Resistances in Heterotrophic, Aerobic Bacteria from Marine Hydrothermal Vent Fields

Chimeric recombinases with designed DNA sequence recognition

Tn3 resolvase catalyses multiple recombination events without intermediate rejoining of DNA ends 1 1 Edited by J. Karn

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