Dynamic stepwise opening of integron attC DNA hairpins by SSB prevents toxicity and ensures functionality.
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ABSTRACT:
SUBMITTER: Grieb MS
PROVIDER: S-EPMC5737091 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Integrons are genetic elements able to integrate and disseminate genes as cassettes by a site-specific recombination mechanism. These elements contain a gene coding for an integrase that carries out recombination by interacting with two different target sites; the attI site in cis with the integrase and the palindromic attC site of a gene cassette. Integron integrases (IntIs) bind specifically to the bottom strand of attC sites. The extrahelical bases resulting from folding of attC bottom strands are important for the recognition by integrases. These enzymes are directly involved in the accumulation and formation of new cassette arrangements in the variable region of integrons. Thus, it is important to better understand interactions between IntIs and their substrates. RESULTS:We compared the ability of five IntIs to carry out excision of several cassettes flanked by different attC sites. The results showed that for most cassettes, IntI1 was the most active integrase. However, IntI2*179E and SonIntIA could easily excise cassettes containing the attCdfrA1 site located upstream, whereas IntI1 and IntI3 had only a weak excision activity for these cassettes. Analysis of the secondary structure adopted by the bottom strand of attCdfrA1 has shown that the identity of the extrahelical bases and the distance between them (A-N7-8-C) differ from those of attCs contained in the cassettes most easily excisable by IntI1 (T-N6-G). We used the attCdfrA1 site upstream of the sat2 gene cassette as a template and varied the identity and spacing between the extrahelical bases in order to determine how these modifications influence the ability of IntI1, IntI2*179E, IntI3 and SonIntIA to excise cassettes. Our results show that IntI1 is more efficient in cassette excision using T-N6-G or T-N6-C attCs while IntI3 recognizes only a limited range of attCs. IntI2*179E and SonIntIA are more tolerant of changes to the identity and spacing of extrahelical bases. CONCLUSIONS:This study provides new insights into the factors that influence the efficiency of cassette excision by integron integrases. It also suggests that IntI2 and SonIntIA have an evolutionary path that is different from IntI1 and IntI3, in their ability to recognize and excise cassettes.
| S-EPMC3053210 | biostudies-literature
Project description:Integrons play a major role in the dissemination of antibiotic resistance genes among bacteria. Rearrangement of gene cassettes occurs by recombination between attI and attC sites, catalyzed by the integron integrase. Integron recombination uses an unconventional mechanism involving a folded single-stranded attC site. This site could be a target for several host factors and more precisely for proteins able to bind single-stranded DNA. One of these, Escherichia coli single-stranded DNA-binding protein (SSB), regulates many DNA processes. We studied the influence of this protein on integron recombination. Our results show the ability of SSB to strongly bind folded attC sites and to destabilize them. This effect was observed only in the absence of the integrase. Indeed, we provided evidence that the integrase is able to counterbalance the observed effect of SSB on attC site folding. We showed that IntI1 possesses an intrinsic property to capture attC sites at the moment of their extrusion, stabilizing them and recombining them efficiently. The stability of DNA secondary structures in the chromosome must be restrained to avoid genetic instability (mutations or deletions) and/or toxicity (replication arrest). SSB, which hampers attC site folding in the absence of the integrase, likely plays an important role in maintaining the integrity and thus the recombinogenic functionality of the integron attC sites. We also tested the RecA host factor and excluded any role of this protein in integron recombination.
| S-EPMC3911172 | biostudies-literature
Project description:Integrons are natural expression vectors in which gene cassettes are integrated downstream of a promoter region by a site-specific recombinase. Gene cassettes usually consist of a single gene followed by a recombination site designated attC. A major unanswered question is how a gene becomes associated with an attC site. Here, we investigate the potential role of a specific lineage of group IIC introns, named group IIC-attC, in cassette formation. Group IIC-attC introns preferentially target attC while retaining the ability to target transcriptional terminators. We show using a PCR-based mobility assay with Escherichia coli that the S.ma.I2 intron from the genome of a clinical isolate of Serratia marcescens can target both attC site and putative terminator motifs of resistance genes. Quantitative results showed that S.ma.I2 is more efficient in targeting various attC sequences than three group IIC-attC introns (54 to 64% sequence identity) from the genomes of environmental isolates. We also show that purified group IIC-attC intron-encoded reverse transcriptases have both RNA-dependent and DNA-dependent DNA polymerase activities in vitro. These data permit us to suggest a new model for gene cassette formation, in which a group IIC-attC intron targets separately a transcriptional terminator adjoining a gene and an isolated attC, joins the gene and the attC by homologous recombination, and then splices and reverse transcribes a gene-attC RNA template, leading to the formation of a cassette.
| S-EPMC2747902 | biostudies-literature
Project description:A predominant tool for adaptation in Gram-negative bacteria is the functional genetic platform called integron. Integrons capture and rearrange promoterless gene cassettes in a unique recombination process involving the recognition of folded single-stranded DNA hairpins-so-called attC sites-with a strong preference for the attC bottom strand. While structural elements have been identified to promote this preference, their mechanistic action remains incomplete. Here, we used high-resolution single-molecule optical tweezers (OT) to characterize secondary structures formed by the attC bottom (${{att}}{{{C}}_{{\rm{bs}}}}$) and top (${{att}}{{{C}}_{{\rm{ts}}}}$) strands of the paradigmatic attCaadA7 site. We found for both sequences two structures-a straight, canonical hairpin and a kinked hairpin. Remarkably, the recombination-preferred ${{att}}{{{C}}_{{\rm{bs}}}}$ predominantly formed the straight hairpin, while the ${{att}}{{{C}}_{{\rm{ts}}}}$ preferentially adopted the kinked structure, which exposes only one complete recombinase binding box. By a mutational analysis, we identified three bases in the unpaired central spacer, which could invert the preferred conformations and increase the recombination frequency of the ${{att}}{{{C}}_{{\rm{ts}}}}$in vivo. A bioinformatics screen revealed structural bias toward a straight, canonical hairpin conformation in the bottom strand of many antibiotic resistance cassettes attC sites. Thus, we anticipate that structural fine tuning could be a mechanism in many biologically active DNA hairpins.
| S-EPMC6393395 | biostudies-literature
Project description:IntroductionFreshwater ecosystems provide propitious conditions for the acquisition and spread of antibiotic resistance genes (ARGs), and integrons play an important role in this process.Material and methodsIn the present study, the diversity of putative environmental integron-cassettes, as well as their potential bacterial hosts in the Velhas River (Brazil), was explored through intI-attC and 16S rRNA amplicons deep sequencing. RESULTS AND DISCUSSION: ORFs related to different biological processes were observed, from DNA integration to oxidation-reduction. ARGs-cassettes were mainly associated with class 1 mobile integrons carried by pathogenic Gammaproteobacteria, and possibly sedentary chromosomal integrons hosted by Proteobacteria and Actinobacteria. Two putative novel ARG-cassettes homologs to fosB3 and novA were detected. Regarding 16SrRNA gene analysis, taxonomic and functional profiles unveiled Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria as dominant phyla. Betaproteobacteria, Alphaproteobacteria, and Actinobacteria classes were the main contributors for KEGG orthologs associated with resistance.ConclusionsOverall, these results provide new information about environmental integrons as a source of resistance determinants outside clinical settings and the bacterial community in the Velhas River.
| S-EPMC7966681 | biostudies-literature
Project description:We recently showed that cassette integration and deletion in integron platforms were occurring through unconventional site-specific recombination reactions involving only the bottom strand of attC sites. The lack of sequence conservation among attC sites led us to hypothesize that sequence-independent structural recognition determinants must exist within attC sites. The structural data obtained from a synaptic complex of the Vibrio cholerae integrase with the bottom strand of an attC site has shown the importance of extra helical bases (EHB) inside the stem-loop structure formed from the bottom strand. Here, we systematically determined the contribution of three structural elements common to all known single-stranded attC site recombination substrates (the EHBs, the unpaired central spacer (UCS), and the variable terminal structure (VTS)) to strand choice and recombination. Their roles have been evaluated in vivo in the attIxattC reaction context using the suicide conjugation assay we previously developed, but also in an attCxattC reaction using a deletion assay. Conjugation was used to deliver the attC sites in single-stranded form. Our results show that strand choice is primarily directed by the first EHB, but the presence of the two other EHBs also serves to increase this strand selection. We found that the structure of the central spacer is essential to achieve high level recombination of the bottom strand, suggesting a dual role for this structure in active site exclusion and for hindering the reverse reaction after the first strand exchange. Moreover, we have shown that the VTS has apparently no role in strand selectivity.
| S-EPMC2727003 | biostudies-literature
Project description:Integrons are bacterial genetic elements that can capture mobile gene cassettes. They are mostly known for their role in the spread of antibiotic resistance cassettes, contributing significantly to the global resistance crisis. These resistance cassettes likely originated from sedentary chromosomal integrons, having subsequently been acquired and disseminated by mobilised integrons. However, their taxonomic and environmental origins are unknown. Here, we use cassette recombination sites (attCs) to predict the origins of those resistance cassettes now spread by mobile integrons. We modelled the structure and sequence homology of 1,978 chromosomal attCs from 11 different taxa. Using these models, we show that at least 27% of resistance cassettes have attCs that are structurally conserved among one of three taxa (Xanthomonadales, Spirochaetes and Vibrionales). Indeed, we found some resistance cassettes still residing in sedentary chromosomal integrons of the predicted taxa. Further, we show that attCs cluster according to host environment rather than host phylogeny, allowing us to assign their likely environmental sources. For example, the majority of β-lactamases and aminoglycoside acetyltransferases, the two most prevalent resistance cassettes, appear to have originated from marine environments. Together, our data represent the first evidence of the taxonomic and environmental origins of resistance cassettes spread by mobile integrons.
| S-EPMC8352920 | biostudies-literature
Project description:The integron platform codes for an integrase (IntI) from the tyrosine family of recombinases that mediates recombination between a proximal double-strand recombination site, attI and a single-strand target recombination site, attC. The attI site is only recognized by its cognate integrase, while the various tested attCs sites are recombined by several different IntI integrases. We have developed a genetic system to enrich and select mutants of IntI1 that provide a higher yield of recombination in order to identify key protein structural elements important for attC x attI1 recombination. We isolated mutants with higher activity on wild type and mutant attC sites. Interestingly, three out of four characterized IntI1 mutants selected on different substrates are mutants of the conserved aspartic acid in position 161. The IntI1 model we made based on the VchIntIA 3D structure suggests that substitution at this position, which plays a central role in multimer assembly, can increase or decrease the stability of the complex and accordingly influence the rate of attI x attC recombination versus attC x attC. These results suggest that there is a balance between the specificity of the protein and the protein/protein interactions in the recombination synapse.
| S-EPMC2095811 | biostudies-other
Project description:Replication protein A (RPA) is an essential eukaryotic single-stranded DNA binding protein with a central role in DNA metabolism. RPA directly participates in DNA double-strand break repair by stimulating 5'-3' end resection by the Sgs1/BLM helicase and Dna2 endonuclease in vitro. Here we investigated the role of RPA in end resection in vivo, using a heat-inducible degron system that allows rapid conditional depletion of RPA in Saccharomyces cerevisiae. We found that RPA depletion eliminated both the Sgs1-Dna2- and Exo1-dependent extensive resection pathways and synergized with mre11? to prevent end resection. The short single-stranded DNA tails formed in the absence of RPA were unstable due to 3' strand loss and the formation of fold-back hairpin structures that required resection initiation and Pol32-dependent DNA synthesis. Thus, RPA is required to generate ssDNA, and also to protect ssDNA from degradation and inappropriate annealing that could lead to genome rearrangements.
| S-EPMC3855855 | biostudies-literature
Project description:The integron is a bacterial recombination system that allows acquisition, stockpiling and expression of cassettes carrying protein-coding sequences, and is responsible for the emergence and rise of multiresistance in Gram-negative bacteria. The functionality of this system depends on the insertion of promoterless cassettes in correct orientation, allowing their expression from the promoter located upstream of the cassette array. Correct orientation is ensured by strand selectivity of integron integrases for the bottom strand of cassette recombination sites (attC), recombined in form of folded single-stranded hairpins. Here, we investigated the basis of such strand selectivity by comparing recombination of wild-type and mutated attC sites with different lengths, sequences and structures. We show that all three unpaired structural features that distinguish the bottom and top strands contribute to strand selectivity. The localization of Extra-Helical Bases (EHBs) directly favors integrase binding to the bottom strand. The Unpaired Central Spacer (UCS) and the Variable Terminal Structure (VTS) influence strand selectivity indirectly, probably through the stabilization of the bottom strand and the resulting synapse due to the nucleotide skew between the two strands. These results underscore the importance of the single-stranded nature of the attC site that allows such tight control over integron cassette orientation.
| S-EPMC5027507 | biostudies-literature