Site-specific accretion of an integrative conjugative element together with a related genomic island leads to cis mobilization and gene capture

ABSTRACT The gene encoding resistance to methicillin and other β-lactam antibiotics in staphylococci, mecA, is carried on a genomic island, SCCmec (for staphylococcal cassette chromosome mec). The chromosomal excision and integration of types I to IV SCCmec are catalyzed by the site-specific recombinases CcrA and CcrB, the genes for which are encoded on each element. We sought to identify the relative contributions of CcrA and CcrB in the excision and integration of SCCmec. Purified CcrB but not CcrA was shown to mediate the gel shift of chromosomal target integration sequences (attB) in electrophoretic mobility shift assays. However, preincubation of CcrB-DNA complexes with increasing concentrations of CcrA blocked gel shift. The interaction of CcrB and CcrA was confirmed by Escherichia coli two-hybrid analysis. SCCmec excision mediated by plasmid-encoded and inducible ccrA, ccrB, or both genes was assessed by PCR in Staphylococcus aureus. CcrB alone could mediate excision but excision was at an alternate att site (attR2) within the right extremity of SCCmec. In contrast, both CcrB and CcrA were required to mediate excision at the chromosomal attB site (called attR when SCCmec is integrated). Insertion of a plasmid containing the SCCmec att site (attS) into the chromosome required both CcrA and CcrB, but CcrA overexpression lowered integration frequency. Thus, while CcrB binds DNA, interaction between CcrA and CcrB, in a precise ratio, is required for attB site-specific excision and SCCmec chromosomal insertion.

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Analysis of RecA-independent recombination events between short direct repeats related to a genomic island and to a plasmid inEscherichia coliK12

PeerJ ◽

10.7717/peerj.3293 ◽

2017 ◽

Vol 5 ◽

pp. e3293 ◽

Cited By ~ 2

Author(s):

María F. Azpiroz ◽

Magela Laviña

Keyword(s):

Genomic Island ◽

Direct Repeat ◽

Genomic Islands ◽

Host Cells ◽

Dna Repeats ◽

Direct Repeats ◽

Site Specific ◽

E Coli ◽

A Site ◽

New Evidence

RecA-independent recombination events between short direct repeats, leading to deletion of the intervening sequences, were found to occur in two genetic models in theEscherichia coliK12 background. The first model was a smallE. coligenomic island which had been shown to be mobile in its strain of origin and, when cloned, also in theE. coliK12 context. However, it did not encode a site-specific recombinase as mobile genomic islands usually do. It was then deduced that the host cells should provide the recombination function. This latter was searched for by means of a PCR approach to detect the island excision inE. coliK12 mutants affected in a number of recombination functions, including the 16E. coliK12 site-specific recombinases, the RecET system, and multiple proteins that participate in the RecA-dependent pathways of homologous recombination. None of these appeared to be involved in the island excision. The second model, analyzed in a RecA deficient context, was a plasmid construction containing a short direct repeat proceeding fromSaccharomyces cerevisiae,which flanked thecatgene. The excision of this gene by recombination of the DNA repeats was confirmed by PCR and through the detection, recovery and characterization of the plasmid deleted form. In sum, we present new evidence on the occurrence of RecA-independent recombination events inE. coliK12. Although the mechanism underlying these processes is still unknown, their existence suggests that RecA-independent recombination may confer mobility to other genetic elements, thus contributing to genome plasticity.

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Analysis of RecA-independent recombination events between short direct repeats related to a genomic island and to a plasmid in Escherichia coli K12

10.7287/peerj.preprints.2807v1 ◽

2017 ◽

Author(s):

María F Azpiroz ◽

Magela D Laviña

Keyword(s):

Escherichia Coli ◽

Genomic Island ◽

Direct Repeat ◽

Host Cells ◽

Dna Repeats ◽

Direct Repeats ◽

Site Specific ◽

E Coli ◽

A Site ◽

New Evidence

RecA-independent recombination events between short direct repeats, leading to deletion of the intervening sequences, were found to occur in two genetic models in the Escherichia coli K12 background. The first model was a small E. coli genomic island which had been shown to be mobile in its strain of origin and, when cloned, in the E. coli K12 context too. However, it did not encode a site-specific recombinase as mobile genomic island usually do. Then, it was deduced that the host cells should provide the recombination function. This latter was searched for by means of a PCR approach to detect the island excision in E. coli K12 mutants affected in a number of recombination functions, including the 16 E. coli K12 site-specific recombinases, the RecET system, and multiple proteins that participate in the RecA-dependent pathways of homologous recombination. None of these appeared to be involved in the island excision. The second model, analyzed in a RecA deficient context, was a plasmid construction containing a short direct repeat proceeding from Saccharomyces cerevisiae, which flanked the cat gene. The excision of this gene by recombination of the DNA repeats was confirmed by PCR and through the detection, recovery and characterization of the plasmid deleted form. In sum, we present new evidence on the occurrence of RecA-independent recombination events in E. coli K12. Although the mechanism underlying these processes is still unknown, their existence suggests that RecA-independent recombination may confer mobility to other genetic elements, thus contributing to genome plasticity.

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Analysis of RecA-independent recombination events between short direct repeats related to a genomic island and to a plasmid in Escherichia coli K12

10.7287/peerj.preprints.2807 ◽

2017 ◽

Author(s):

María F Azpiroz ◽

Magela D Laviña

Keyword(s):

Escherichia Coli ◽

Genomic Island ◽

Direct Repeat ◽

Host Cells ◽

Dna Repeats ◽

Direct Repeats ◽

Site Specific ◽

E Coli ◽

A Site ◽

New Evidence

RecA-independent recombination events between short direct repeats, leading to deletion of the intervening sequences, were found to occur in two genetic models in the Escherichia coli K12 background. The first model was a small E. coli genomic island which had been shown to be mobile in its strain of origin and, when cloned, in the E. coli K12 context too. However, it did not encode a site-specific recombinase as mobile genomic island usually do. Then, it was deduced that the host cells should provide the recombination function. This latter was searched for by means of a PCR approach to detect the island excision in E. coli K12 mutants affected in a number of recombination functions, including the 16 E. coli K12 site-specific recombinases, the RecET system, and multiple proteins that participate in the RecA-dependent pathways of homologous recombination. None of these appeared to be involved in the island excision. The second model, analyzed in a RecA deficient context, was a plasmid construction containing a short direct repeat proceeding from Saccharomyces cerevisiae, which flanked the cat gene. The excision of this gene by recombination of the DNA repeats was confirmed by PCR and through the detection, recovery and characterization of the plasmid deleted form. In sum, we present new evidence on the occurrence of RecA-independent recombination events in E. coli K12. Although the mechanism underlying these processes is still unknown, their existence suggests that RecA-independent recombination may confer mobility to other genetic elements, thus contributing to genome plasticity.

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Characterization of a Novel Integrative Element, ICESt1, in the Lactic Acid BacteriumStreptococcus thermophilus

Applied and Environmental Microbiology ◽

10.1128/aem.66.4.1749-1753.2000 ◽

2000 ◽

Vol 66 (4) ◽

pp. 1749-1753 ◽

Cited By ~ 27

Author(s):

Vincent Burrus ◽

Yvonne Roussel ◽

Bernard Decaris ◽

Gérard Guédon

Keyword(s):

Lactic Acid ◽

Streptococcus Thermophilus ◽

Conjugative Transfer ◽

Gene Encoding ◽

Conjugative Transposon ◽

Site Specific ◽

Conjugative Transposons ◽

Integrative Conjugative Element ◽

A Site

ABSTRACT The 35.5-kb ICESt1 element of Streptococcus thermophilus CNRZ368 is bordered by a 27-bp repeat and integrated into the 3′ end of a gene encoding a putative fructose-1,6-biphosphate aldolase. This element encodes site-specific integrase and excisionase enzymes related to those of conjugative transposons Tn5276and Tn5252. The integrase was found to be involved in a site-specific excision of a circular form. ICESt1 also encodes putative conjugative transfer proteins related to those of the conjugative transposon Tn916. Therefore, ICESt1could be or could be derived from an integrative conjugative element.

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Large-cluster and combined fluorescent and gold immunoprobes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166920 ◽

1996 ◽

Vol 54 ◽

pp. 892-893

Author(s):

Richard D. Powell ◽

James F. Hainfeld ◽

Carol M. R. Halsey ◽

David L. Spector ◽

Shelley Kaurin ◽

...

Keyword(s):

Metal Cluster ◽

Sulfonyl Chloride ◽

Gel Filtration ◽

Cross Linking ◽

Site Specific ◽

Fab Fragments ◽

Cluster Label ◽

Covalently Linked ◽

Texas Red ◽

Fold Excess

Two new types of covalently linked, site-specific immunoprobes have been prepared using metal cluster labels, and used to stain components of cells. Combined fluorescein and 1.4 nm “Nanogold” labels were prepared by using the fluorescein-conjugated tris (aryl) phosphine ligand and the amino-substituted ligand in the synthesis of the Nanogold cluster. This cluster label was activated by reaction with a 60-fold excess of (sulfo-Succinimidyl-4-N-maleiniido-cyclohexane-l-carboxylate (sulfo-SMCC) at pH 7.5, separated from excess cross-linking reagent by gel filtration, and mixed in ten-fold excess with Goat Fab’ fragments against mouse IgG (obtained by reduction of F(ab’)2 fragments with 50 mM mercaptoethylamine hydrochloride). Labeled Fab’ fragments were isolated by gel filtration HPLC (Superose-12, Pharmacia). A combined Nanogold and Texas Red label was also prepared, using a Nanogold cluster derivatized with both and its protected analog: the cluster was reacted with an eight-fold excess of Texas Red sulfonyl chloride at pH 9.0, separated from excess Texas Red by gel filtration, then deprotected with HC1 in methanol to yield the amino-substituted label.

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The challenge of detecting modifications on proteins

Essays in Biochemistry ◽

10.1042/ebc20190055 ◽

2020 ◽

Vol 64 (1) ◽

pp. 135-153 ◽

Cited By ~ 1

Author(s):

Lauren Elizabeth Smith ◽

Adelina Rogowska-Wrzesinska

Keyword(s):

Mass Spectrometry ◽

Protein Function ◽

Chemical Properties ◽

Lysine Acetylation ◽

Mass Determination ◽

Post Translational Modifications ◽

Site Specific ◽

The Common

Abstract Post-translational modifications (PTMs) are integral to the regulation of protein function, characterising their role in this process is vital to understanding how cells work in both healthy and diseased states. Mass spectrometry (MS) facilitates the mass determination and sequencing of peptides, and thereby also the detection of site-specific PTMs. However, numerous challenges in this field continue to persist. The diverse chemical properties, low abundance, labile nature and instability of many PTMs, in combination with the more practical issues of compatibility with MS and bioinformatics challenges, contribute to the arduous nature of their analysis. In this review, we present an overview of the established MS-based approaches for analysing PTMs and the common complications associated with their investigation, including examples of specific challenges focusing on phosphorylation, lysine acetylation and redox modifications.

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