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Karuppannan Veluthambi - One of the best experts on this subject based on the ideXlab platform.
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generation of selectable marker free sheath blight resistant transgenic rice plants by efficient co transformation of a Cointegrate vector t dna and a binary vector t dna in one agrobacterium tumefaciens strain
Plant Cell Reports, 2008Co-Authors: Rajasekaran Sripriya, Vengoji Raghupathy, Karuppannan VeluthambiAbstract:Co-transformation of Oryza sativa L. var. Pusa Basmati1 was done using an Agrobacteriumtumefaciens strain harbouring a single-copy Cointegrate vector and a multi-copy binary vector in the same cell. The T-DNA of the Cointegrate vector pGV2260::pSSJ1 carried the hygromycin phosphotransferase (hph) and β-glucuronidase (gus) genes. The binary vector pCam-chi11, without a plant selectable marker gene, harboured the rice chitinase (chi11) gene under maize ubiquitin promoter. Co-transformation of the gene of interest (chi11) with the selectable marker gene (hph) occurred in 4 out of 20 T0 plants (20%). Segregation of hph from chi11 was accomplished in two (CoT6 and CoT23) of the four co-transformed plants in the T1 generation. The selectable marker-free (SMF) lines CoT6 and CoT23 harboured single copies of chi11. Homozygous SMF T2 plants were established in the lines CoT6 and CoT23. Northern and Western blot analysis of the homozygous SMF lines showed high level of transgene expression. In comparison to untransformed controls, chitinase specific activity was 66- and 22-fold higher in the homozygous SMF T2 plants of lines CoT6 and CoT23, respectively. The lines CoT6 and CoT23 exhibited 38 and 40% reduction in sheath blight disease, respectively.
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Generation of selection marker-free transgenic plants by cotransformation of a Cointegrate vector T-DNA and a binary vector T-DNA in one Agrobacterium tumefaciens strain
Plant Science, 2002Co-Authors: Subha S. Jacob, Karuppannan VeluthambiAbstract:Cotransformation of Nicotiana tabacum was done using a single Agrobacterium tumefaciens strain harbouring a Cointegrate vector (one copy per cell) and a binary vector (ten to 15 copies per cell). The T-DNAs of the Cointegrate plasmid, pGV2260::pSSJ1 and the binary plasmid, pGA472 carried hph and nptII as plant selection markers, respectively. When the binary T-DNA marker (nptII) was used for selection, the non-selected Cointegrate T-DNA with hph cotransformed at 36% frequency. However, upon using the Cointegrate T-DNA with hph for selection, cotransformation of binary T-DNA with nptII was much higher (56-74%). Segregation of the T-DNA markers hph and nptII was followed in the T 1 generation to screen for the elimination of the selection marker. The elimination of nptII of the multicopy binary vector was found in the progeny of only one of the three T 0 plants and at a low frequency of 3%. However, elimination of hph of the single-copy Cointegrate vector was found at 16-18% frequency in the progeny of both the T 0 plants analysed. The use of the T-DNA of low copy number Cointegrate vector for initial selection improves the cotransformation frequency of non-selected T-DNA of the multicopy binary vector. This strategy also increases the frequency of generation of selection marker-free transgenic plants in the T 1 generation.
Ruth M Hall - One of the best experts on this subject based on the ideXlab platform.
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targeted conservative Cointegrate formation mediated by is26 family members requires sequence identity at the reacting end
mSphere, 2021Co-Authors: Christopher J Harmer, Ruth M HallAbstract:ABSTRACT IS26 forms Cointegrates using two distinct routes, a copy-in mechanism involving one insertion sequence (IS) and a target and a targeted conservative mechanism involving two ISs in different DNA molecules. In this study, the ability of IS26 and some close relatives, IS1006, IS1008, and a natural hybrid, IS1006/IS1008, which are found predominantly in Acinetobacter spp., to interact was examined. IS1006/1008 consists of 175 bp from IS1006 at the left end, with the remainder from IS1008. These ISs all have the same 14-bp terminal inverted repeats, and the Tnp26, Tnp1006, and Tnp1008 transposases, with pairwise identities of 83.7% to 93.1%, should be able to recognize each other’s ends. In a recA-negative Escherichia coli strain, IS1006, IS1008, and IS1006/1008 each formed Cointegrates via the copy-in route and via the targeted conservative route, albeit at frequencies for the targeted reaction at least 10-fold lower than for IS26. However, using mixed pairs, targeted cointegration was detected only when IS1008 was paired with the IS1006/1008 hybrid, which also encodes Tnp1008, and the targeted Cointegrates formed all arose from a reaction occurring at the end where the DNA sequences are identical. The reaction also occurred at the end with extended DNA identity using IS26 paired with IS26::catA1, an artificially constructed IS26 derivative that includes the catA1 gene. Thus, both identical transposases and identical DNA sequences at the reacting end were required. These features indicate that the targeted conservative pathway proceeds via a single transposase-catalyzed strand transfer, followed by migration and resolution of the Holliday junction formed. IMPORTANCE The IS26 family includes the ISs that are commonly found associated with antibiotic resistance genes in multiply resistant Gram-negative and Gram-positive bacteria. IS26 is most prevalent in Gram-negative species and can generate the clusters of antibiotic resistance genes interspersed with directly oriented IS26 seen in multiply resistant pathogens. This ability relies on the novel dual mechanistic capabilities of IS26 family members. However, the mechanism underlying the recently discovered targeted conservative mode of Cointegrate formation mediated by IS26, IS257/IS431, and IS1216, which is unlike any previously studied IS movement mechanism, is not well understood. An important question is what features of the IS and the transposase are key to allowing IS26 family members to undertake targeted conservative reaction. In this study, this question was addressed using mixed-partner crosses involving IS26 and naturally occurring close relatives of IS26 that are found near resistance genes in Acinetobacter baumannii and are widespread in Acinetobacter species.
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structures bounded by directly oriented members of the is26 family are pseudo compound transposons
Plasmid, 2020Co-Authors: Christopher J Harmer, Carol H Pong, Ruth M HallAbstract:Antibiotic resistance genes are often found in structures bounded by copies of IS26, IS257/IS431 or IS1216 that resemble compound (or composite) transposons. However, because of the mechanisms used by IS26 family members, namely that they form Cointegrates but cannot resolve them, none of these structures can move together as a coherent single unit. Apparent transposition of these structures is possible via a 2-step process but only if the IS are in direct orientation. An intermolecular reaction catalysed by the IS-encoded transposase and an intramolecular homologous recombination step can occur in either order. In one route, one of the IS bounding the structure forms a Cointegrate between the DNA molecule carrying it and a target molecule. Cointegrates formed by either copy-in or targeted conservative routes contain three directly-oriented IS copies and can be resolved by homologous recombination between specific pairs of IS, with one pair leading to apparent transposition of the whole structure. In the other route, homologous recombination first forms a circular intermediate, a translocatable unit or TU, which is incorporated by the transposase either at a random site or adjacent to another IS copy in a target molecule. We therefore conclude that the transposon-like structures are not compound (or composite) transposons and the nomenclature for them should be revised. We propose that the term "pseudo compound transposon" (PCT), first coined in 1989, should be used to describe those structures where the IS are in direct orientation. Structures with the IS in opposite orientation should not be named as transposons.
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IS26 Family Members IS257 and IS1216 Also Form Cointegrates by Copy-In and Targeted Conservative Routes
'American Society for Microbiology', 2020Co-Authors: Christopher J Harmer, Ruth M HallAbstract:ABSTRACT IS26 has been shown to form Cointegrates both by a copy-in mechanism involving one insertion sequence (IS) and a target and by a targeted conservative mechanism involving two ISs. IS26 is the flagship of a group of 65 bacterial ISs in the recently redefined IS6/IS26 family. Here, whether other family members can also use two mechanisms was examined using members of the IS257/IS431 and IS1216 isoform groups, which are associated with antibiotic resistance genes in staphylococci and enterococci, respectively. Transposases Tnp257 and Tnp1216 have 39% and 47% amino acid identities, respectively, with Tnp26 and are 62% identical to one another. Using a novel transposition assay, pUC-based plasmids carrying these ISs integrated into the chromosome of a temperature-sensitive polA Escherichia coli strain grown at the restrictive temperature. In the Cointegrates, the plasmid carrying IS257 was flanked by various 8-bp target site duplications, consistent with random target selection. However, in a mating-out assay, only the targeted conservative reaction was detectable at a low frequency in a recA-negative E. coli strain, indicating that IS257 is at least 100-fold less active than IS26. For IS1216, in mating-out assays, both copy-in and targeted conservative Cointegrate formation were detectable at frequencies similar to those observed for IS26. Duplication of various 8-bp target sites was detected for the copy-in route. For both IS257 and IS1216, when both of the plasmids carried an IS, the targeted conservative route occurred at a significantly higher frequency than the copy-in route, and only Cointegrates formed by the conservative route were detected. IMPORTANCE IS26 differs from other studied ISs in the reactions that it can undertake. The differences make IS26 uniquely suited to its key role in the recruitment and spread of antibiotic resistance genes in Gram-negative bacteria. However, whether other ISs in the IS6/IS26 family can perform the same reactions is not known. IS257/IS431 and IS1216 isoforms found associated with antibiotic resistance genes in the Gram-positive bacteria staphylococci, enterococci, streptococci, and clostridia are related to IS26. However, the way that they move had not been investigated, limiting interpretation of their role in resistance gene dissemination and in the formation of Cointegrates and complex resistance regions in staphylococci and enterococci. Here, they are shown to share the broad catalytic capabilities of IS26, demonstrating that it is likely that all members of the redefined IS6/IS26 family of bacterial ISs likewise are able to use both the copy-in and conservative routes
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Movement of IS26-associated antibiotic resistance genes occurs via a translocatable unit that includes a single IS26 and preferentially inserts adjacent to another IS26. MBio 2014
2016Co-Authors: Christopher J Harmer, Robert A. Moran, Ruth M HallAbstract:ABSTRACT The insertion sequence IS26 plays a key role in disseminating antibiotic resistance genes in Gram-negative bacteria, forming regions containing more than one antibiotic resistance gene that are flanked by and interspersed with copies of IS26. A model presented for a secondmode of IS26movement that explains the structure of these regions involves a translocatable unit consisting of a unique DNA segment carrying an antibiotic resistance (or other) gene and a single IS copy. Structures resembling class I transposons are generated via RecA-independent incorporation of a translocatable unit next to a second IS26 such that the ISs are in direct orientation. Repeating this process would lead to arrays of resistance genes with directly oriented copies of IS26 at each end and between each unique segment. This model requires that IS26 recognizes another IS26 as a target, and in transpo-sition experiments, the frequency of Cointegrate formation was 60-fold higher when the target plasmid contained IS26. This re-action was conservative, with no additional IS26 or target site duplication generated, and orientation specific as the IS26s in the Cointegrates were always in the same orientation. Consequently, the Cointegrates were identical to those formed via the known mode of IS26movement when a target IS26was not present. Intact transposase genes in both IS26s were required for high-frequency Cointegrate formation as inactivation of either one reduced the frequency 30-fold. However, the IS26 target specificity was retained. Conversion of each residue in the DDEmotif of the Tnp26 transposase also reduced the cointegration frequency. IMPORTANCE Resistance to antibiotics belonging to several of the different classes used to treat infections is a critical problem. Multiply antibiotic-resistant bacteria usually carry large regions containing several antibiotic resistance genes, and in Gram
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movement of is26 associated antibiotic resistance genes occurs via a translocatable unit that includes a single is26 and preferentially inserts adjacent to another is26
Mbio, 2014Co-Authors: Christopher J Harmer, Robert A. Moran, Ruth M HallAbstract:ABSTRACT The insertion sequence IS 26 plays a key role in disseminating antibiotic resistance genes in Gram-negative bacteria, forming regions containing more than one antibiotic resistance gene that are flanked by and interspersed with copies of IS 26 . A model presented for a second mode of IS 26 movement that explains the structure of these regions involves a translocatable unit consisting of a unique DNA segment carrying an antibiotic resistance (or other) gene and a single IS copy. Structures resembling class I transposons are generated via RecA-independent incorporation of a translocatable unit next to a second IS 26 such that the ISs are in direct orientation. Repeating this process would lead to arrays of resistance genes with directly oriented copies of IS 26 at each end and between each unique segment. This model requires that IS 26 recognizes another IS 26 as a target, and in transposition experiments, the frequency of Cointegrate formation was 60-fold higher when the target plasmid contained IS 26 . This reaction was conservative, with no additional IS 26 or target site duplication generated, and orientation specific as the IS 26 s in the Cointegrates were always in the same orientation. Consequently, the Cointegrates were identical to those formed via the known mode of IS 26 movement when a target IS 26 was not present. Intact transposase genes in both IS 26 s were required for high-frequency Cointegrate formation as inactivation of either one reduced the frequency 30-fold. However, the IS 26 target specificity was retained. Conversion of each residue in the DDE motif of the Tnp26 transposase also reduced the cointegration frequency. IMPORTANCE Resistance to antibiotics belonging to several of the different classes used to treat infections is a critical problem. Multiply antibiotic-resistant bacteria usually carry large regions containing several antibiotic resistance genes, and in Gram-negative bacteria, IS 26 is often seen in these clusters. A model to explain the unusual structure of regions containing multiple IS 26 copies, each associated with a resistance gene, was not available, and the mechanism of their formation was unexplored. IS 26 -flanked structures deceptively resemble class I transposons, but this work reveals that the features of IS 26 movement do not resemble those of the IS and class I transposons studied to date. IS 26 uses a novel movement mechanism that defines a new family of mobile genetic elements that we have called “translocatable units.” The IS 26 mechanism also explains the properties of IS 257 (IS 431 ) and IS 1216 , which belong to the same IS family and mobilize resistance genes in Gram-positive staphylococci and enterococci.
Philip Hans Franses - One of the best experts on this subject based on the ideXlab platform.
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forecasting the levels of vector autoregressive log transformed time series
International Journal of Forecasting, 2000Co-Authors: Miguel A Arino, Philip Hans FransesAbstract:In this paper we give explicit expressions for the forecasts of levels of a vector time series when such forecasts are generated from (possibly Cointegrated) vector autoregressions for the corresponding log-transformed time series. We also show that simply taking exponentials of forecasts for logged data leads to substantially biased forecasts. We illustrate this using a bivariate Cointegrated vector series containing US GNP and investments.
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forecasting the levels of vector autoregressive log transformed time series
Econometric Institute Research Papers, 1996Co-Authors: Miguel A Arino, Philip Hans FransesAbstract:textabstractIn this paper we give explicit expressions for the forecasts of levels of a vector time series when such forecasts are generated from (possibly Cointegrated) vector autoregressions for the corresponding log-transformed time series. We also show that simply taking exponentials of forecasts for logged data leads to substantially biased forecasts. We illustrate this using a bivariate Cointegrated vector series containing US GNP and investments.
Matthew K Waldor - One of the best experts on this subject based on the ideXlab platform.
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genomic and functional analysis of icepdaspa1 a fish pathogen derived sxt related integrating conjugative element that can mobilize a virulence plasmid
Journal of Bacteriology, 2008Co-Authors: Carlos R Osorio, Joeli Marrero, Rachel A F Wozniak, Manuel L Lemos, Vincent Burrus, Matthew K WaldorAbstract:Integrating conjugative elements (ICEs) are self-transmissible mobile elements that transfer between bacteria via conjugation and integrate into the host chromosome. SXT and related ICEs became prevalent in Asian Vibrio cholerae populations in the 1990s and play an important role in the dissemination of antibiotic resistance genes in V. cholerae. Here, we carried out genomic and functional analyses of ICEPdaSpa1, an SXT-related ICE derived from a Spanish isolate of Photobacterium damselae subsp. piscicida, the causative agent of fish pasteurellosis. The approximately 102-kb DNA sequence of ICEPdaSpa1 shows nearly 97% DNA sequence identity to SXT in genes that encode essential ICE functions, including integration and excision, conjugal transfer, and regulation. However, approximately 25 kb of ICEPdaSpa1 DNA, including a tetracycline resistance locus, is not present in SXT. Most ICEPdaSpa1-specific DNA is inserted at loci where other SXT-related ICEs harbor element-specific DNA. ICEPdaSpa1 excises itself from the chromosome and is transmissible to other Photobacterium strains, as well as to Escherichia coli, in which it integrates into prfC. Interestingly, the P. damselae virulence plasmid pPHDP10 could be mobilized from E. coli in an ICEPdaSpa1-dependent fashion via the formation of a Cointegrate between pPHDP10 and ICEPdaSpa1. pPHDP10-Cm integrated into ICEPdaSpa1 in a non-site-specific fashion independently of RecA. The ICEPdaSpa1::pPHDP10 Cointegrates were stable, and markers from both elements became transmissible at frequencies similar to those observed for the transfer of ICEPdaSpa1 alone. Our findings reveal the plasticity of ICE genomes and demonstrate that ICEs can enable virulence gene transfer.
Christopher J Harmer - One of the best experts on this subject based on the ideXlab platform.
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targeted conservative Cointegrate formation mediated by is26 family members requires sequence identity at the reacting end
mSphere, 2021Co-Authors: Christopher J Harmer, Ruth M HallAbstract:ABSTRACT IS26 forms Cointegrates using two distinct routes, a copy-in mechanism involving one insertion sequence (IS) and a target and a targeted conservative mechanism involving two ISs in different DNA molecules. In this study, the ability of IS26 and some close relatives, IS1006, IS1008, and a natural hybrid, IS1006/IS1008, which are found predominantly in Acinetobacter spp., to interact was examined. IS1006/1008 consists of 175 bp from IS1006 at the left end, with the remainder from IS1008. These ISs all have the same 14-bp terminal inverted repeats, and the Tnp26, Tnp1006, and Tnp1008 transposases, with pairwise identities of 83.7% to 93.1%, should be able to recognize each other’s ends. In a recA-negative Escherichia coli strain, IS1006, IS1008, and IS1006/1008 each formed Cointegrates via the copy-in route and via the targeted conservative route, albeit at frequencies for the targeted reaction at least 10-fold lower than for IS26. However, using mixed pairs, targeted cointegration was detected only when IS1008 was paired with the IS1006/1008 hybrid, which also encodes Tnp1008, and the targeted Cointegrates formed all arose from a reaction occurring at the end where the DNA sequences are identical. The reaction also occurred at the end with extended DNA identity using IS26 paired with IS26::catA1, an artificially constructed IS26 derivative that includes the catA1 gene. Thus, both identical transposases and identical DNA sequences at the reacting end were required. These features indicate that the targeted conservative pathway proceeds via a single transposase-catalyzed strand transfer, followed by migration and resolution of the Holliday junction formed. IMPORTANCE The IS26 family includes the ISs that are commonly found associated with antibiotic resistance genes in multiply resistant Gram-negative and Gram-positive bacteria. IS26 is most prevalent in Gram-negative species and can generate the clusters of antibiotic resistance genes interspersed with directly oriented IS26 seen in multiply resistant pathogens. This ability relies on the novel dual mechanistic capabilities of IS26 family members. However, the mechanism underlying the recently discovered targeted conservative mode of Cointegrate formation mediated by IS26, IS257/IS431, and IS1216, which is unlike any previously studied IS movement mechanism, is not well understood. An important question is what features of the IS and the transposase are key to allowing IS26 family members to undertake targeted conservative reaction. In this study, this question was addressed using mixed-partner crosses involving IS26 and naturally occurring close relatives of IS26 that are found near resistance genes in Acinetobacter baumannii and are widespread in Acinetobacter species.
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structures bounded by directly oriented members of the is26 family are pseudo compound transposons
Plasmid, 2020Co-Authors: Christopher J Harmer, Carol H Pong, Ruth M HallAbstract:Antibiotic resistance genes are often found in structures bounded by copies of IS26, IS257/IS431 or IS1216 that resemble compound (or composite) transposons. However, because of the mechanisms used by IS26 family members, namely that they form Cointegrates but cannot resolve them, none of these structures can move together as a coherent single unit. Apparent transposition of these structures is possible via a 2-step process but only if the IS are in direct orientation. An intermolecular reaction catalysed by the IS-encoded transposase and an intramolecular homologous recombination step can occur in either order. In one route, one of the IS bounding the structure forms a Cointegrate between the DNA molecule carrying it and a target molecule. Cointegrates formed by either copy-in or targeted conservative routes contain three directly-oriented IS copies and can be resolved by homologous recombination between specific pairs of IS, with one pair leading to apparent transposition of the whole structure. In the other route, homologous recombination first forms a circular intermediate, a translocatable unit or TU, which is incorporated by the transposase either at a random site or adjacent to another IS copy in a target molecule. We therefore conclude that the transposon-like structures are not compound (or composite) transposons and the nomenclature for them should be revised. We propose that the term "pseudo compound transposon" (PCT), first coined in 1989, should be used to describe those structures where the IS are in direct orientation. Structures with the IS in opposite orientation should not be named as transposons.
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IS26 Family Members IS257 and IS1216 Also Form Cointegrates by Copy-In and Targeted Conservative Routes
'American Society for Microbiology', 2020Co-Authors: Christopher J Harmer, Ruth M HallAbstract:ABSTRACT IS26 has been shown to form Cointegrates both by a copy-in mechanism involving one insertion sequence (IS) and a target and by a targeted conservative mechanism involving two ISs. IS26 is the flagship of a group of 65 bacterial ISs in the recently redefined IS6/IS26 family. Here, whether other family members can also use two mechanisms was examined using members of the IS257/IS431 and IS1216 isoform groups, which are associated with antibiotic resistance genes in staphylococci and enterococci, respectively. Transposases Tnp257 and Tnp1216 have 39% and 47% amino acid identities, respectively, with Tnp26 and are 62% identical to one another. Using a novel transposition assay, pUC-based plasmids carrying these ISs integrated into the chromosome of a temperature-sensitive polA Escherichia coli strain grown at the restrictive temperature. In the Cointegrates, the plasmid carrying IS257 was flanked by various 8-bp target site duplications, consistent with random target selection. However, in a mating-out assay, only the targeted conservative reaction was detectable at a low frequency in a recA-negative E. coli strain, indicating that IS257 is at least 100-fold less active than IS26. For IS1216, in mating-out assays, both copy-in and targeted conservative Cointegrate formation were detectable at frequencies similar to those observed for IS26. Duplication of various 8-bp target sites was detected for the copy-in route. For both IS257 and IS1216, when both of the plasmids carried an IS, the targeted conservative route occurred at a significantly higher frequency than the copy-in route, and only Cointegrates formed by the conservative route were detected. IMPORTANCE IS26 differs from other studied ISs in the reactions that it can undertake. The differences make IS26 uniquely suited to its key role in the recruitment and spread of antibiotic resistance genes in Gram-negative bacteria. However, whether other ISs in the IS6/IS26 family can perform the same reactions is not known. IS257/IS431 and IS1216 isoforms found associated with antibiotic resistance genes in the Gram-positive bacteria staphylococci, enterococci, streptococci, and clostridia are related to IS26. However, the way that they move had not been investigated, limiting interpretation of their role in resistance gene dissemination and in the formation of Cointegrates and complex resistance regions in staphylococci and enterococci. Here, they are shown to share the broad catalytic capabilities of IS26, demonstrating that it is likely that all members of the redefined IS6/IS26 family of bacterial ISs likewise are able to use both the copy-in and conservative routes
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Movement of IS26-associated antibiotic resistance genes occurs via a translocatable unit that includes a single IS26 and preferentially inserts adjacent to another IS26. MBio 2014
2016Co-Authors: Christopher J Harmer, Robert A. Moran, Ruth M HallAbstract:ABSTRACT The insertion sequence IS26 plays a key role in disseminating antibiotic resistance genes in Gram-negative bacteria, forming regions containing more than one antibiotic resistance gene that are flanked by and interspersed with copies of IS26. A model presented for a secondmode of IS26movement that explains the structure of these regions involves a translocatable unit consisting of a unique DNA segment carrying an antibiotic resistance (or other) gene and a single IS copy. Structures resembling class I transposons are generated via RecA-independent incorporation of a translocatable unit next to a second IS26 such that the ISs are in direct orientation. Repeating this process would lead to arrays of resistance genes with directly oriented copies of IS26 at each end and between each unique segment. This model requires that IS26 recognizes another IS26 as a target, and in transpo-sition experiments, the frequency of Cointegrate formation was 60-fold higher when the target plasmid contained IS26. This re-action was conservative, with no additional IS26 or target site duplication generated, and orientation specific as the IS26s in the Cointegrates were always in the same orientation. Consequently, the Cointegrates were identical to those formed via the known mode of IS26movement when a target IS26was not present. Intact transposase genes in both IS26s were required for high-frequency Cointegrate formation as inactivation of either one reduced the frequency 30-fold. However, the IS26 target specificity was retained. Conversion of each residue in the DDEmotif of the Tnp26 transposase also reduced the cointegration frequency. IMPORTANCE Resistance to antibiotics belonging to several of the different classes used to treat infections is a critical problem. Multiply antibiotic-resistant bacteria usually carry large regions containing several antibiotic resistance genes, and in Gram
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movement of is26 associated antibiotic resistance genes occurs via a translocatable unit that includes a single is26 and preferentially inserts adjacent to another is26
Mbio, 2014Co-Authors: Christopher J Harmer, Robert A. Moran, Ruth M HallAbstract:ABSTRACT The insertion sequence IS 26 plays a key role in disseminating antibiotic resistance genes in Gram-negative bacteria, forming regions containing more than one antibiotic resistance gene that are flanked by and interspersed with copies of IS 26 . A model presented for a second mode of IS 26 movement that explains the structure of these regions involves a translocatable unit consisting of a unique DNA segment carrying an antibiotic resistance (or other) gene and a single IS copy. Structures resembling class I transposons are generated via RecA-independent incorporation of a translocatable unit next to a second IS 26 such that the ISs are in direct orientation. Repeating this process would lead to arrays of resistance genes with directly oriented copies of IS 26 at each end and between each unique segment. This model requires that IS 26 recognizes another IS 26 as a target, and in transposition experiments, the frequency of Cointegrate formation was 60-fold higher when the target plasmid contained IS 26 . This reaction was conservative, with no additional IS 26 or target site duplication generated, and orientation specific as the IS 26 s in the Cointegrates were always in the same orientation. Consequently, the Cointegrates were identical to those formed via the known mode of IS 26 movement when a target IS 26 was not present. Intact transposase genes in both IS 26 s were required for high-frequency Cointegrate formation as inactivation of either one reduced the frequency 30-fold. However, the IS 26 target specificity was retained. Conversion of each residue in the DDE motif of the Tnp26 transposase also reduced the cointegration frequency. IMPORTANCE Resistance to antibiotics belonging to several of the different classes used to treat infections is a critical problem. Multiply antibiotic-resistant bacteria usually carry large regions containing several antibiotic resistance genes, and in Gram-negative bacteria, IS 26 is often seen in these clusters. A model to explain the unusual structure of regions containing multiple IS 26 copies, each associated with a resistance gene, was not available, and the mechanism of their formation was unexplored. IS 26 -flanked structures deceptively resemble class I transposons, but this work reveals that the features of IS 26 movement do not resemble those of the IS and class I transposons studied to date. IS 26 uses a novel movement mechanism that defines a new family of mobile genetic elements that we have called “translocatable units.” The IS 26 mechanism also explains the properties of IS 257 (IS 431 ) and IS 1216 , which belong to the same IS family and mobilize resistance genes in Gram-positive staphylococci and enterococci.