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PubMed1996

The Bacteroides mobilizable insertion element, NBU1, integrates into the 3' end of a Leu-tRNA gene and has an integrase that is a member of the lambda integrase family.

NBU1 is a 10.3-kbp integrated Bacteroides element that can be induced to excise from the chromosome and can be mobilized to a recipient by trans-acting functions provided by certain Bacteroides conjugative transposons. The NBU1 transfer intermediate is a covalently closed circle, which is presumed to be the form that integrates into the recipient genome. We report here that a 2.4-kbp segment of NBU1 was all that was required for site-specific integration into the chromosome of Bacteroides thetaiotaomicron 5482. This 2.4-kbp region included the joined ends of the NBU1 circular form (attN1) and a single open reading frame, intN1, which encoded the integrase. Previously, we had found that NBU1 integrates preferentially into a single site in B. thetaiotaomicron 5482. We have now shown that the NBU1 target site is located at the 3' end of a Leu-tRNA gene. The NBU1 integrase gene, intN1, was sequenced. The predicted protein had little overall amino acid sequence similarity to any proteins in the databases but had limited carboxy-terminal similarity to the integrases of lambdoid phages and to the integrases of the gram-positive conjugative transposons Tn916 and Tn1545. We also report that the intN1 gene is expressed constitutively.

Amino Acid Sequence↗
PubMed2006

The current ICE age: biology and evolution of SXT-related integrating conjugative elements.

SXT is an integrating conjugative element (ICE) that was initially isolated from a 1992 Vibrio cholerae O139 clinical isolate from India. This approximately 100-kb ICE encodes resistance to multiple antibiotics. SXT or closely related ICEs are now present in most clinical and some environmental V. cholerae isolates from Asia and Africa. SXT-related ICEs are not limited to V. cholerae. It is now clear that so-called IncJ elements such as R391 are closely related to SXT. More than 25 members of the SXT/R391 family of ICEs have now been identified in environmental and clinical isolates of diverse species of gamma-proteobacteria worldwide. In this review, we discuss the diversity, evolution and biology of this family of ICEs.

Bacterial Proteins↗
PubMed2000

Multiple gene products and sequences required for excision of the mobilizable integrated Bacteroides element NBU1.

NBU1 is an integrated 10.3-kbp Bacteroides element, which can excise and transfer to Bacteroides or Escherichia coli recipients, where it integrates into the recipient genome. NBU1 relies on large, >60-kbp, conjugative transposons for factors that trigger excision and for mobilization of the circular form to recipients. Previously, we showed that a single integrase gene, intN1, was necessary and sufficient for integration of NBU1 into its target site on the Bacteroides or E. coli genome. We now show that an unexpectedly large region of NBU1 is required for excision. This region includes, in addition to intN1, four open reading frames plus a large region downstream of the fourth gene, prmN1. This downstream sequence was designated XRS, for "excision-required sequence." XRS contains the oriT of the circular form of NBU1 and about two-thirds of the adjacent mobilization gene, mobN1. This is the first time an oriT, which is involved in conjugal transfer of the circular form, has been implicated in excision. Disruption of the gene immediately downstream of intN1, orf2, completely abolished excision. The next open reading frame, orf2x, was too small to be disrupted, so we still do not know whether it plays a role in the excision reaction. Deletions were made in each of two open reading frames downstream of orf2x, orf3 and prmN1. Both of these deletions abolished excision, indicating that these genes are also essential for excision. Attempts to complement various mutations in the excision region led us to realize that a portion of the excision region carrying prmN1 and part of the XRS (XRS(HIII)) inhibited excision when provided in trans on a multicopy plasmid (8 to 10 copies per cell). However, a fragment carrying prmN1, XRS, and the entire mobilization gene, mobN1, did not have this effect. The smaller fragment may be interfering with excision by attracting proteins made by the intact NBU1 and thus removing them from the excision complex. Our results show clearly that excision is a complex process that involves several proteins and a cis-acting region (XRS) which includes the oriT. We suggest that this complex excision machinery may be necessary to allow NBU1 to coordinate nicking at the ends during excision and nicking at the oriT during conjugal transfer, to prevent premature nicking at the oriT before NBU1 has excised and circularized.

Bacterial Proteins↗
PubMed1999

The site-specific integration of genetic elements may modulate thermostable protease production, a virulence factor in Dichelobacter nodosus, the causative agent of ovine footrot.

The gram-negative anaerobe Dichelobacter nodosus is the causative agent of footrot in sheep. The authors have previously characterized two genetic elements, the intA (vap) and intB elements, which integrate into the genome of D. nodosus. In the virulent strain A198 there are two copies of the intA element. One copy is integrated into the 3' end of the tRNA-serGCU gene, close to the aspartokinase (askA) gene, and the second copy is integrated into the 3' end of the tRNA-serGGA gene, next to the polynucleotide phosphorylase (pnpA) gene. In this study, a new genetic element was identified in the benign strain C305, the intC element, integrated into the 3' end of the tRNA-serGCU gene, next to askA. The intC element was found in most D. nodosus strains, both benign and virulent, which were examined, and was integrated into tRNA-serGCU in most strains. Between the askA and tRNA-serGCU genes, a gene (designated glpA), was identified whose predicted protein product has very high amino acid identity with RsmA from the plant pathogen Erwinia carotovora. RsmA acts as a global repressor of pathogenicity in E. carotovora, by repressing the production of extracellular enzymes. In virulent strains of D. nodosus the intA element was found to be integrated next to pnpA, and either the intA or intC element was integrated next to glpA. By contrast, all but one of the benign strains had intB at one or both of these two positions, and the one exception had neither intA, intB nor intC at one position. The loss of the intC element from the virulent strain 1311 resulted in loss of thermostable protease activity, a virulence factor in D. nodosus. A model for virulence is proposed whereby integration of the intA and intC genetic elements modulates virulence by altering the expression of glpA, pnpA, tRNA-serGCU and tRNA-serGGA.

3' Untranslated Regions↗
PubMed1995

The mobilization regions of two integrated Bacteroides elements, NBU1 and NBU2, have only a single mobilization protein and may be on a cassette.

Bacteroides conjugative transposons can act in trans to excise, circularize, and transfer unlinked integrated elements called NBUs (for nonreplicating Bacteroides units). Previously, we localized and sequenced the mobilization region of one NBU, NBU1, and showed that this mobilization region was recognized by the IncP plasmids RP4 and R751, as well as by the Bacteroides conjugative transposons. We report here that the single mobilization protein carried by NBU1 appears to be a bifunctional protein that binds to the oriT region and catalyzes the nicking reaction that initiates the transfer process. We have also localized and sequenced the mobilization region of a second NBU, NBU2. The NBU2 mobilization region was 86 to 90% identical at the DNA sequence to the oriT-mob region of NBU1. The high sequence similarity between NBU1 and NBU2 ended abruptly after the stop codon of the mob gene and about 1 kbp upstream of the oriT region, indicating that the oriT-mob regions of NBU1 and NBU2 may be on some sort of cassette. A region on NBU1 and NBU2 which lies immediately upstream of the oriT region had 66% sequence identity to a region upstream of the oriT region on a mobilizable transposon, Tn4399, an element that had previously appeared to be completely unrelated to the NBUs.

Amino Acid Sequence↗
PubMed2004

Archaeal integrases and mechanisms of gene capture.

Archaeal integrases facilitate the formation of two distinctive types of integrated element within archaeal chromosomes: the SSV type and pNOB8 type. The former carries a smaller N-terminal and a larger C-terminal integrase gene fragment, and the latter an intact integrase gene. All integrated elements overlap tRNA genes that were target sites for integration. It has been demonstrated that SSV (Sulfolobus spindle virus) viruses, carrying an SSV-type integrase gene, and conjugative plasmids, carrying a pNOB8-type integrase, are integrative elements. Two mechanisms have been proposed for stably maintaining an integrated element within archaeal chromosomes. There is also evidence for changes having occurred in the captured integrated elements present in archaeal genomes. Thus we infer that site-specific integration constitutes an important mechanism for horizontal gene transfer and genome evolution.

Archaea↗
PubMed2006

SXT-related integrating conjugative element in New World Vibrio cholerae.

SXT-related integrating conjugative elements (ICEs) became prevalent in Asian Vibrio cholerae populations after V. cholerae O139 emerged. Here, we describe an SXT-related ICE, ICEVchMex1, in a Mexican environmental V. cholerae isolate. Identification of ICEVchMex1 represents the first description of an SXT-related ICE in the Western Hemisphere. The significant differences between the SXT and ICEVchMex1 genomes suggest that these ICEs have evolved independently.

Conjugation, Genetic↗
PubMed2025

Global diversity of integrating conjugative elements (ICEs) in Helicobacter pylori and their influence on genome architecture.

Integrating conjugative elements (ICEs) are mobile genetic elements conferring a wide range of beneficial functions upon their bacterial hosts. Generally, they can be activated from their integrated states to undergo horizontal gene transfer via conjugation. In the case of the human gastric pathogen Helicobacter pylori, a paradigm for extensive genetic diversity, highly efficient natural transformation and recombination processes may superimpose canonical transfer of its two ICEs termed ICEHptfs3 and ICEHptfs4, and thus shape their composition substantially. Here, as a part of the Helicobacter pylori Genome Project (HpGP) initiative, we have analyzed high-quality genome sequences from 1011 clinical strains with respect to their ICE content and variability. We show that both elements are highly prevalent in all H. pylori populations, but have a strong tendency for gene erosion. ICE sequence variations reflect the population structure and show a clear signature of increased horizontal transfer. A detailed map of ICE integration sites revealed local preferences, but also how recombination processes result in hybrid elements or genome rearrangements. Population-specific differences in ICE cargo genes might reflect distinct requirements in the biological functions provided by these mobile elements.

Journal Article↗
PubMed2004

The large resolvase TnpX is the only transposon-encoded protein required for transposition of the Tn4451/3 family of integrative mobilizable elements.

Chloramphenicol resistance in Clostridium perfringens and Clostridium difficile is often encoded by catP genes located within the 6.3 kb integrative mobilizable elements Tn4451 and Tn4453 respectively. This family of transposons is capable of being mobilized into a recipient cell in the presence of another conjugative element. Transposition is mediated by the large resolvase TnpX, which excises the element to produce a circular molecule that is the integrative intermediate. In this study, in vivo deletion analysis of the transposon-encoded tnpV and tnpY genes showed that they are not essential for excision or integration of this group of elements. Similar studies on tnpW suggested either that this gene is not essential for these functions or that TnpW does not function when provided in trans. Development and use of an in vivo insertion assay showed that TnpX is the only transposon-encoded protein required for the integration reaction. Subsequently, a TnpXLEH6 protein was purified and shown to catalyse excision in vitro in the absence of any other protein and preferentially to excise a supercoiled DNA substrate. In summary, these studies have shown that TnpX is the only transposon protein required in vivo and in vitro for the excision process and that, like excision, integration also occurs by a serine recombinase-mediated site-specific recombination mechanism.

Chloramphenicol Resistance↗
PubMed2001

Dynamic signaling between astrocytes and neurons.

Astrocytes, a sub-type of glia in the central nervous system, are dynamic signaling elements that integrate neuronal inputs, exhibit calcium excitability, and can modulate neighboring neurons. Neuronal activity can lead to neurotransmitter-evoked activation of astrocytic receptors, which mobilizes their internal calcium. Elevations in astrocytic calcium in turn trigger the release of chemical transmitters from astrocytes, which can cause sustained modulatory actions on neighboring neurons. Astrocytes, and perisynaptic Schwann cells, by virtue of their intimate association with synapses, are strategically positioned to regulate synaptic transmission. This capability, that has now been demonstrated in several studies, raises the untested possibility that astrocytes are an integral element of the circuitry for synaptic plasticity. Because the highest ratio of glia-to-neurons is found at the top of the phylogenetic tree in the human brain, these recent demonstrations of dynamic bi-directional signaling between astrocytes and neurons leave us with the question as to whether astrocytes are key regulatory elements of higher cortical functions.

Animals↗
PubMed1982

Nucleotide sequence analysis of avian retroviruses: structural similarities with transposable elements.

Integrated retroviral genomes are flanked by direct repeats of sequences derived from the termini of the viral RNA genome. These sequences are designated long terminal repeats (LTRs). We have determined and analyzed the nucleotide sequence of the LTRs from several exogenous and endogenous avian retroviruses. These LTRs possess several structural similarities with eukaryotic and prokaryotic transposable elements: 1) inverted complementary repeats at the termini, 2) deletions of sequences adjacent to the LTR, 3) small duplications of host sequences flanking the integrated provirus, and 4) sequence homologies with transposable and other genetic elements. These observations suggest that LTRs function in the integration and perhaps transposition of retrovirus genomes. Evidence exists for the presence of a strong promoter sequence within the LTR. The retroviral LTR also contains a "Hogness box" up-stream of the capping site and a poly(A) signal. These features suggest an additional role for the LTR in the regulation of gene expression.

Animals↗
PubMed1998

Int-B13, an unusual site-specific recombinase of the bacteriophage P4 integrase family, is responsible for chromosomal insertion of the 105-kilobase clc element of Pseudomonas sp. Strain B13.

Pseudomonas sp. strain B13 carries the clcRABDE genes encoding chlorocatechol-degradative enzymes on the self-transmissible 105-kb clc element. The element integrates site and orientation specifically into the chromosomes of various bacterial recipients, with a glycine tRNA structural gene (glyV) as the integration site. We report here the localization and nucleotide sequence of the integrase gene and the activity of the integrase gene product in mediating site-specific integration. The integrase gene (int-B13) was located near the right end of the clc element. It consisted of an open reading frame (ORF) of maximally 1,971 bp with a coding capacity for 657 amino acids (aa). The full-length protein (74 kDa) was observed upon overexpression and sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation. The N-terminal 430 aa of the predicted Int-B13 protein had substantial similarity to integrases from bacteriophages of the P4 family, but Int-B13 was much larger than P4-type integrases. The C-terminal 220 aa of Int-B13 were homologous to an ORF flanking a gene cluster for naphthalene degradation in Pseudomonas aeruginosa PaK1. Similar to the bacteriophages phiR73 and P4, the clc element integrates into the 3' end of the target tRNA gene. This target site was characterized from four different recipient strains into which the clc element integrated, showing sequence specificity of the integration. In Pseudomonas sp. strain B13, a circular form of the clc element, which carries an 18-bp DNA sequence identical to the 3'-end portion of glyV as part of its attachment site (attP), could be detected. Upon chromosomal integration of the clc element into a bacterial attachment site (attB), a functional glyV was reconstructed at the right end of the element. The integration process could be demonstrated in RecA-deficient Escherichia coli with two recombinant plasmids, one carrying the int-B13 gene and the attP site and the other carrying the attB site of Pseudomonas putida F1.

Amino Acid Sequence↗
PubMed1997

Construction and characterization of a Bacteroides thetaiotaomicron recA mutant: transfer of Bacteroides integrated conjugative elements is RecA independent.

We report the construction and analysis of a Bacteroides thetaiotaomicron recA disruption mutant and an investigation of whether RecA is required for excision and integration of Bacteroides mobile DNA elements. The recA mutant was deficient in homologous recombination and was more sensitive than the wild-type strain to DNA-damaging agents. The recA mutant was also more sensitive to oxygen than the wild type, indicating that repair of DNA contributes to the aerotolerance of B. thetaiotaomicron. Many Bacteroides clinical isolates carry self-transmissible chromosomal elements known as conjugative transposons. These conjugative transposons can also excise and mobilize in trans a family of unlinked integrated elements called nonreplicating Bacteroides units (NBUs). The results of a previous study had raised the possibility that RecA plays a role in excision of Bacteroides conjugative transposons, but this hypothesis could not be tested in Bacteroides spp. because no RecA-deficient Bacteroides strain was available. We report here that the excision and integration of the Bacteroides conjugative transposons, as well as NBU1 and Tn4351, were unaffected by the absence of RecA activity.

Aerobiosis↗
PubMed2003

piggyBac-based insertional mutagenesis in the presence of stably integrated P elements in Drosophila.

P element-mediated mutagenesis has been used to disrupt an estimated 25% of genes essential for Drosophila adult viability. Mutation of all genes in the fly genome, however, poses a problem, because P elements show significant hotspots of integration. In addition, advanced screening scenarios often require the use of P element-based tools like the generation of germ-line mosaics using FLP recombinase-mediated recombination or gene misexpression using the UAS/Gal4 system. These techniques are P element-based and can therefore not be combined with the use of P elements as mutagenic agents. To circumvent these limitations, we have developed an insertional mutagenesis system using non-P element transposons. An enhanced yellow fluorescent protein-marked piggyBac-based mutator element was mobilized by a piggyBac specific transposase source expressed from a Hermes-based jump-starter transposon marked with enhanced cyan fluorescent protein. In a pilot screen, we have generated 798 piggyBac insertions on FRT bearing third chromosomes of which 9% have sustained a putatively piggyBac-related lethal hit. The FRTs present on the target chromosome remained stably integrated during the screen and could subsequently be used to generate germ-line clones associated with maternal and zygotic phenotypes. PCR-based analysis of insertion loci shows that 57% of the insertions are in genes for which no P element insertions have been reported. Our data demonstrate the potential of this technique to facilitate the quest for saturation mutagenesis of the Drosophila genome. The system is Drosophila nonspecific and potentially applicable in a broad spectrum of nonmodel organisms.

Animals↗
PubMed2000

Potential gene conversion and source genes for recently integrated Alu elements.

Alu elements comprise >10% of the human genome. We have used a computational biology approach to analyze the human genomic DNA sequence databases to determine the impact of gene conversion on the sequence diversity of recently integrated Alu elements and to identify Alu elements that were potentially retroposition competent. We analyzed 269 Alu Ya5 elements and identified 23 members of a new Alu subfamily termed Ya5a2 with an estimated copy number of 35 members, including the de novo Alu insertion in the NF1 gene. Our analysis of Alu elements containing one to four (Ya1-Ya4) of the Ya5 subfamily-specific mutations suggests that gene conversion contributed as much as 10%-20% of the variation between recently integrated Alu elements. In addition, analysis of the middle A-rich region of the different Alu Ya5 members indicates a tendency toward expansion of this region and subsequent generation of simple sequence repeats. Mining the databases for putative retroposition-competent elements that share 100% nucleotide identity to the previously reported de novo Alu insertions linked to human diseases resulted in the retrieval of 13 exact matches to the NF1 Alu repeat, three to the Alu element in BRCA2, and one to the Alu element in FGFR2 (Apert syndrome). Transient transfections of the potential source gene for the Apert's Alu with its endogenous flanking genomic sequences demonstrated the transcriptional and presumptive transpositional competency of the element.

Alleles↗
PubMed2005

Molecular tools to detect the IncJ elements: a family of integrating, antibiotic resistant mobile genetic elements.

The IncJ group of enterobacterial mobile genetic elements, which include R391, R392, R705, R997 and pMERPH, have been shown to be site-specific integrating elements encoding variable antibiotic and heavy metal resistance genes. They insert into a specific 17-bp site located in the prfC gene, encoding peptide release factor 3, in Escherichia coli and other hosts. A key feature of known IncJ elements is the presence of a site-specific recombination module consisting of an attachment site on the element and an integrase-encoding gene of the tyrosine recombinase class, which promotes integration between the attachment site on the element and a similar site on the host chromosome. We have cloned and sequenced the integrases from a number of known IncJ elements and designed PCR primers for specific amplification of this gene. Using conserved regions of enterobacterial prfC genes upstream and downstream of the insertion site, and conserved sequences at the ends of the integrated IncJ elements, we have designed specific primers to amplify across the integrated IncJ attL and attR junction fragments. Alignment of over 30 enterobacterial prfC-like genes indicates that the primers designed to amplify attR junction would amplify IncJ element: host junctions from a wide variety of hosts. The IncJ elements have been shown to sensitise recA(+)E. coli K12 strains to UV irradiation. A simple and rapid procedure for demonstrating this effect is described. These tools should enable the rapid detection of such elements in clinical and environmental settings.

Attachment Sites, Microbiological↗
PubMed1995

Conjugative transposons: an unusual and diverse set of integrated gene transfer elements.

Conjugative transposons are integrated DNA elements that excise themselves to form a covalently closed circular intermediate. This circular intermediate can either reintegrate in the same cell (intracellular transposition) or transfer by conjugation to a recipient and integrate into the recipient's genome (intercellular transposition). Conjugative transposons were first found in gram-positive cocci but are now known to be present in a variety of gram-positive and gram-negative bacteria also. Conjugative transposons have a surprisingly broad host range, and they probably contribute as much as plasmids to the spread of antibiotic resistance genes in some genera of disease-causing bacteria. Resistance genes need not be carried on the conjugative transposon to be transferred. Many conjugative transposons can mobilize coresident plasmids, and the Bacteroides conjugative transposons can even excise and mobilize unlinked integrated elements. The Bacteroides conjugative transposons are also unusual in that their transfer activities are regulated by tetracycline via a complex regulatory network.

Anti-Bacterial Agents↗