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PubMed1994

Identification of an HP1 phage protein required for site-specific excision.

Transposon insertion mutagenesis and transformation were used to locate genes responsible for excision in the temperature phage HP1 of Haemophilus influenzae. A 6.5 kb segment of DNA near the left end of the phage genome was sequenced, and 11 new open reading frames were identified. Two face-to-face overlapping promoter sequences organized these open reading frames into two operons transcribed in opposite directions. Interruption of the first open reading frame in the rightward operon created lysogens unable to produce phages. Provision of the uninterrupted open reading frame in trans restored phage production. The gene identified by this procedure, cox, was cloned and the protein product was expressed at high levels in Escherichia coli. The Cox protein is a 79-residue basic protein with a predicted strong helix-turn-helix DNA-binding motif. Extracts induced to express high levels of Cox contained a 9 kDa protein. These extracts inhibited integrative recombination and were required for excisive recombination mediated by HP1 integrase. The HP1 cox gene location is similar to that of the homologous excisive and regulatory genes from coliphages P2 and 186. These phages appear to share a distinctive organization of recombination proteins and transcriptional domains differing markedly from phage lambda and its relatives.

Amino Acid Sequence↗
PubMed1994

The sss gene product, which affects pyoverdin production in Pseudomonas aeruginosa 7NSK2, is a site-specific recombinase.

Pyoverdin production by Pseudomonas aeruginosa strain 7NSK2 was induced by Zn(II) in the presence of iron. A mutant was isolated in which Zn(II) no longer induced pyoverdin production. The sss gene which was inactivated in this mutant was cloned and sequenced. Its protein sequence showed 50% identity to the XerC protein of Escherichia coli, which is a member of the lambda integrase family of site-specific recombinases. An open reading frame was found upstream of sss whose protein sequence showed strong identity to DapF, the diaminopimelate epimerase. In E. coli, xerC is part of a multicistronic unit that also contains dapF. The sss gene of P. aeruginosa could restore site-specific recombination at cer in an E. coli xerC mutant and the E. coli xerC gene could complement a genomic sss mutation in P. aeruginosa.

Amino Acid Sequence↗
PubMed1995

Plasmid evolution by acquisition of mobile gene cassettes: plasmid pIE723 contains the aadB gene cassette precisely inserted at a secondary site in the incQ plasmid RSF1010.

Gene cassettes are mobile DNA elements which contain a specific recombination site, a 59-base element, recognized by the site-specific recombination system of integrons. Gene cassettes are normally found inserted at a unique site in an integron, downstream of a promoter which directs transcription of the cassette-associated genes. However, insertion of a gene cassette into a secondary site in a plasmid which does not contain an integron is also formally possible. Sequence analysis of the aadB gene in pIE723, a plasmid closely related to the IncQ plasmid RSF1010, revealed the presence of the complete aadB cassette inserted at a secondary site downstream of a known RSF1010 promoter. The site of insertion of the aadB cassette in RSF1010 conformed to the consensus for secondary sites recognized by the integron integrase (Int), and it is likely that the cassette was inserted via a single Int-mediated recombination event between the 59-base element of a free, circular aadB cassette and a secondary site in RSF1010. The cassette-associated recombination site was inactivated by the insertion, and Int-mediated excision of the aadB cassette from this non-specific location was not detectable, indicating that the cassette is stably inserted. The movement of gene cassettes to secondary sites is likely to play an important role in the acquisition of new genes by bacterial and plasmid genomes.

Amino Acid Sequence↗
PubMed1995

Novel organization of the site-specific integration and excision recombination functions of the Staphylococcus aureus serotype F virulence-converting phages phi 13 and phi 42.

Functions required for site-specific integration and excision of the Staphylococcus aureus serotype F virulence-converting phages phi 13 and phi 42 were localized and characterized. Like other temperate phages, integration of phi 13 and phi 42 sequences was found to require the product of an int gene located close to the phage attP site. Both int genes are almost identical, express proteins possessing characteristic features of the Int (integrase) family of recombinases, but share very little homology with previously described int genes, including those of the serotype B S. aureus phages L54a and phi 11. Nevertheless, all four S. aureus phages share an almost identical short sequence located immediately 5' to these distinct int genes, suggesting a common mechanism of int gene regulation. Upstream from these common sequences, the sequences of phi 13 and phi 42 are quite distinct from each other, and from the corresponding regions of phi 11 and L54a which encode the Xis proteins that are required with Int to mediate site-specific excision of the latter phages. Surprisingly, phi 13 and phi 42 sequences encompassing the attP sites and int genes, but lacking either an adjacent or more distant phage excision protein gene, were sufficient to mediate site-specific excision of integrated phage DNA sequences.

Amino Acid Sequence↗
PubMed1995

Molecular genetics of the chloramphenicol-resistance transposon Tn4451 from Clostridium perfringens: the TnpX site-specific recombinase excises a circular transposon molecule.

The chloramphenicol-resistance transposon Tn4451 undergoes precise conjugative deletion from its parent plasmid plP401 in Clostridium perfringens and precise spontaneous excision from multicopy plasmids in Escherichia coli. The complete nucleotide sequence of the 6338 bp transposon was determined and it was found to encode six genes. Genetic analysis demonstrated that the largest Tn4451-encoded gene, tnpX, was required for the spontaneous excision of the transposon in both E. coli and C. perfringens, since a Tn4451 derivative that lacked a functional tnpX gene was completely stable in both organisms. Because the ability of this derivative to excise was restored by providing the tnpX gene on a compatible plasmid, it was concluded that this gene encoded a trans-acting site-specific recombinase. Allelic exchange was used to introduce the tnpX delta 1 allele onto plP401 and it was shown that TnpX was also required for the conjugative excision of Tn4451 in C. perfringens. It was also shown by hybridization and polymerase chain reaction (PCR) studies that TnpX-mediated transposon excision resulted in the formation of a circular form of the transposon. The TnpX recombinase was unique because it potentially contained the motifs of two independent site-specific recombinase families, namely the resolvase/invertase and integrase families. Sequence analysis indicated that the resolvase/invertase domain of TnpX was likely to be involved in the excision process by catalysing the formation of a 2 bp staggered nick on either side of the GA dinucleotide located at the ends of the transposon and at the junction of the circular form. The other Tn4451-encoded genes include tnpZ, which appears to encode a second potential site-specific recombinase. This protein has similarity to plasmid-encoded Mob/Pre proteins, which are involved in plasmid mobilization and multimer formation. Located upstream of the tnpZ gene was a region with similarity to the site of interaction of these mobilization proteins.

Amino Acid Sequence↗
PubMed2004

Characterization of XerC- and XerD-dependent CTX phage integration in Vibrio cholerae.

CTXphi is a filamentous bacteriophage that encodes cholera toxin and integrates site-specifically into the larger of the two Vibrio cholerae chromosomes. The CTXphi genome lacks an integrase; instead, its integration depends on the chromosome-encoded tyrosine recombinases XerC and XerD. During integration, recombination occurs between regions of homology in CTXphi and the V. cholerae chromosome. Here, we define the elements on the phage genome (attP) and bacterial chromosome (attB) required for CTXphi integration. attB is a short sequence composed of one binding site for XerC and XerD spanning the site of recombination. Together, XerC and XerD bind to two sites within attP. While one XerC/D binding site in attP spans the core recombination region, the other site is approximately 80 bp away. Although integration occurs at the core XerC/D binding site in attP, the second site is required for CTXphi integration, suggesting it performs an architectural role in the integration reaction. In vitro cleavage reactions showed that XerC and XerD are capable of cleaving attB and attP sequences; however, additional cellular processes such as DNA replication or Holliday junction resolution by a host resolvase may contribute to integration in vivo.

Base Sequence↗
PubMed1997

Cloning and analysis of the glucosyl transferase gene encoding type I antigen in Shigella flexneri.

The O-antigen of most Shigella flexneri serotypes contains an identical tetrasaccharide repeating unit. Apart from serotype Y, the O-antigen is modified by addition of a glucosyl and/or O-acetyl residue to a specific position in the O-unit. In this study the glucosyl transferase gene from a serotype 1 a has been cloned and identified. The bacteriophage SfV integrase (int) gene was used to probe a S. flexneri Y53 (serotype 1 a) cosmid library and 18 unique clones were identified. Southern hybridisation of these clones indicated two unlinked regions of the chromosome contained the int homologue. When expressed in a live candidate vaccine strain of S. flexneri serotype Y (SFL124), clones with one region produced type I antigen, whereas clones containing the other region produced mainly type Y antigen. One of the cosmid clones positive for type I antigen by agglutination and Western blotting was selected for further study. Genes involved in O-antigen glucosyl modification were mapped on a 5.8 kb fragment and subclones were produced which fully or partially expressed the type I antigen, depending on the extent of the clone. Fully and partially expressing clones may be useful vaccine candidate strains for protection against disease caused by two serotypes of S. flexneri.

Antigens, Bacterial↗
PubMed1998

Cloning and characterisation of the Proteus mirabilis xerD gene.

The Xer site-specific recombination system is involved in the stable maintenance of replicons (certain plasmids and chromosomes) in Escherichia coli and other bacteria by converting multimers into monomers. This system requires a cis-acting DNA sequence (the chromosomal dif site or the ColE1 cer site) and two trans-acting factors: the XerC and XerD recombinases, which belong to the lambda integrase family of tyrosine site-specific recombinases. In addition, in order to resolve plasmid multimers into monomers, two additional factors are required: the ArgR and PepA proteins. We have previously shown the presence of xerC and xerD genes (and their function) by Southern hybridisation and by in vivo recombination in a wide variety of Enterobacteriaceac. We have now cloned and sequenced the xerD gene of Proteus mirabilis using degenerate and inverse PCR methods. This gene encodes a tyrosine recombinase which is highly similar to the E. coli XerD recombinase, is capable of complementing an E. coli xerD mutant, and displays sequence-specific DNA binding activity.

Amino Acid Sequence↗
PubMed1998

Serotype conversion of a Shigella flexneri candidate vaccine strain via a novel site-specific chromosome-integration system.

Shigella flexneri SFL124 (serotype Y) is a promising live oral vaccine candidate, which has been shown to be safe and immunogenic in human volunteers. To change the serotype of this vaccine strain, we inserted a serotype conversion gene cluster into the chromosome of SFL124 by using a bacteriophage-based site-specific integration system. By cloning an integrase gene (int), an attachment site (attP) and a glucosyl transfer gene cluster from bacteriophage SfX into a suicide vector, and subsequently introducing this construct into S. flexneri SFL124, we obtained a S. flexneri strain (designated SFL1213) expressing the serotype X somatic antigen specificity. The strain retained other characteristics of the parent strain, such as colony shape, growth rate, and Congo red binding property. Stability test showed that the serotype X O-antigen specificity in SFL1213 was 100% stable after being cultured approximately 72 successive hours under non-selective condition. In a mouse pulmonary model, the recombinant strain elicited a significant level of humoral antibodies which recognized the lipopolysaccharide (LPS) of a wild-type S. flexneri serotype X strain. The site-specific insertion system will be useful when stable expression of a cloned single copy gene is desired in the chromosome of S. flexneri vaccine candidate, SFL124.

Animals↗
PubMed2001

Molecular heterogeneity of bla(VIM-2)-containing integrons from Pseudomonas aeruginosa plasmids encoding the VIM-2 metallo-beta-lactamase.

A bla(VIM-2) metallo-beta-lactamase determinant, identical to that previously identified in Pseudomonas aeruginosa COL-1 isolate from a French hospital, was detected on a 28-kb plasmid carried by a nosocomial isolate of P. aeruginosa from Verona, Italy. In this plasmid the bla(VIM-2) determinant was inserted into a class 1 integron of original structure, named In72, that contains a partially deleted intI1 integrase gene and two gene cassettes. The first cassette carries an aacA4 aminoglycoside acetyl transferase determinant. The second cassette carries a bla(VIM-2) determinant followed by a partially deleted attC site. The structure of In72 was notably different from that of In56, the bla(VIM-2)-containing integron found in the COL-1 isolate, revealing the existence of molecular heterogeneity among bla(VIM-2)-containing integrons in clinical isolates of P. aeruginosa from Europe.

Acetyltransferases↗
PubMed2002

Mobile elements in archaeal genomes.

The recent availability of several archaeal genome sequences has provided a basis for detailed analyses of the frequency, location and phylogeny of archaeal mobile elements. All the known elements fall into two main types, autonomous insertion sequence (IS) elements and the non-autonomous miniature inverted repeat element (MITE)-like elements. Both classes are considered to be mobilized via transposases that are encoded by the IS elements, although mobility has only been demonstrated experimentally for a few elements. The number, and diversity, of the elements differs greatly between the genomes. At one extreme Sulfolobus solfataricus P2 and Halobacterium NRC-1 are very rich in elements while Methanobacterium thermoautotrophicum contains none. The former also show examples of complex clusters of interwoven elements. An analysis of the genomic distribution in S. solfataricus suggests that the putative oriC and terC regions act as barriers for the mobility of both IS and MITE-like elements. Moreover, the very high level of truncated IS elements in the genomes of S. solfataricus, Sulfolobus tokodaii and Thermoplasma volcanium suggests that there may be a cellular mechanism for selectively inactivating IS elements at a point when they become too numerous and disadvantageous for the cell. Phylogenetically, archaeal IS elements are confined to 11 of the 17 known families of bacterial and eukaryal IS elements where some generate distinct subgroups. Finally, DNA viruses, plasmids and DNA fragments can also be inserted into, and excised from, archaeal genomes by means of an integrase-mediated mechanism that has special archaeal characteristics.

Crenarchaeota↗
PubMed2006

Analysis of integrons in clinical isolates of Escherichia coli in China during the last six years.

A multiple PCR for the detection of the integrase genes of the three classes of integrons was carried out, and their gene cassettes were characterized in 111 clinical strains of Escherichia coli isolated in Guangzhou City, China during the last 6 years. IntI1 and intI2 genes were detected in 95 isolates (85.6%) and four isolates (3.6%), respectively. No intI3 gene was detected. Six different gene cassettes were found in these strains, and a high prevalence of dfr and aad genes was observed. The E. coli isolates that contained a 1664-bp amplicon of dfrA17-aadA5 in class 1 integron were found to be phylogenetically unrelated to each other by using the enterobacterial repetitive intergenic consensus PCR, as the cassette could be transferred to recipient strains, indicating that the gene cassettes might be disseminated in the clinical strains by a horizontal gene transfer. Therefore, it is important that guidelines for the prudent use of antimicrobial agents are adopted and surveillance programs are established.

Anti-Bacterial Agents↗
PubMed2006

A novel ICE in the genome of Shewanella putrefaciens W3-18-1: comparison with the SXT/R391 ICE-like elements.

A novel R391-like ICE (integrating conjugative element) has been detected in the 4.2 MB genome of Shewanella putrefaciens W3-18-1 located on three different contigs. Assembly of the ICE encoding contigs based on similarity with R391 revealed a mosaic element of plasmid, phage and transposon-like sequences typical of SXT/R391 ICE-like elements. The element, which is 110 057 bp in length, was highly similar to R391 sequences, with most related ORFs showing >96% amino acid sequence identity. The element, designated ICESpuPO1, contained a number of inserts determining resistance to copper and other heavy metals and a broad-spectrum RND efflux pump similar to antibiotic efflux systems. The element was integrated into the Shewanella prfC gene in a manner similar to related ICE-like elements. The chromosomal element junctions contained a 17-bp SXT/R391-like attL and attR site and an unannotated ORF between attL and the ICE integrase encoding a putative recombinational directional factor necessary for excision, with 100% amino acid identity to the R391 ORF4 product.

Bacterial Proteins↗
PubMed2006

Kinetic study of the HIV-1 DNA 3'-end processing.

The 3'-processing of viral DNA extremities is the first step in the integration process catalysed by human immunodeficiency virus (HIV)-1 integrase (IN). This reaction is relatively inefficient and processed DNAs are usually detected in vitro under conditions of excess enzyme. Despite such experimental conditions, steady-state Michaelis-Menten formalism is often applied to calculate characteristic equilibrium/kinetic constants of IN. We found that the amount of processed product was not significantly affected under conditions of excess DNA substrate, indicating that IN has a limited turnover for DNA cleavage. Therefore, IN works principally in a single-turnover mode and is intrinsically very slow (single-turnover rate constant = 0.004 min(-1)), suggesting that IN activity is mainly limited at the chemistry step or at a stage that precedes chemistry. Moreover, fluorescence experiments showed that IN-DNA product complexes were very stable over the time-course of the reaction. Binding isotherms of IN to DNA substrate and product also indicate tight binding of IN to the reaction product. Therefore, the slow cleavage rate and limited product release prevent or greatly reduce subsequent turnover. Nevertheless, the time-course of product formation approximates to a straight line for 90 min (apparent initial velocity), but we show that this linear phase is due to the slow single-turnover rate constant and does not indicate steady-state multiple turnover. Finally, our data ruled out the possibility that there were large amounts of inactive proteins or dead-end complexes in the assay. Most of complexes initially formed were active although dramatically slow.

DNA, Viral↗
PubMed1998

Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA.

Topoisomerases I promote the relaxation of DNA superhelical tension by introducing a transient single-stranded break in duplex DNA and are vital for the processes of replication, transcription, and recombination. The crystal structures at 2.1 and 2.5 angstrom resolution of reconstituted human topoisomerase I comprising the core and carboxyl-terminal domains in covalent and noncovalent complexes with 22-base pair DNA duplexes reveal an enzyme that "clamps" around essentially B-form DNA. The core domain and the first eight residues of the carboxyl-terminal domain of the enzyme, including the active-site nucleophile tyrosine-723, share significant structural similarity with the bacteriophage family of DNA integrases. A binding mode for the anticancer drug camptothecin is proposed on the basis of chemical and biochemical information combined with these three-dimensional structures of topoisomerase I-DNA complexes.

Antineoplastic Agents, Phytogenic↗
PubMed1998

A distinctive class of integron in the Vibrio cholerae genome.

The ability of bacteria to acquire and disseminate heterologous genes has been a major factor in the development of multiple drug resistance. A gene, intI4, was identified that encodes a previously unknown integrase that is associated with a "gene-VCR" organization (VCRs are Vibrio cholerae repeated sequences), similar to that of the well-characterized antibiotic resistance integrons. The similarity was confirmed by IntI1-mediated recombination of a gene-VCR cassette into a class 1 integron. VCR cassettes are found in a number of Vibrio species including a strain of V. metschnikovii isolated in 1888, suggesting that this mechanism of heterologous gene acquisition predated the antibiotic era.

Amino Acid Sequence↗
PubMed1995

Requirement of RNA polymerase III transcription factors for in vitro position-specific integration of a retroviruslike element.

The yeast retroviruslike element Ty3 inserts at the transcription initiation sites of genes transcribed by RNA polymerase III (Pol III). An in vitro integration assay was developed with the use of Ty3 viruslike particles and a modified SUP2 tyrosine transfer RNA (tRNA(Tyr)) gene target. Integration was position-specific and required Ty3 integrase, Pol III transcription factor (TF) IIIB-, TFIIIC-, and Pol III-containing fractions showed that TFIIIB and TFIIIC, together, were sufficient for position-specific Ty3 integration, but not for transcription. This report demonstrates that in vitro integration of a retroelement can be targeted by cellular proteins.

DNA Nucleotidyltransferases↗
PubMed1999

Reconstruction of an active integron recombination site after integration of a gene cassette at a secondary site.

As the site of insertion of the aadB gene cassette on pRAY, from a clinical isolate of Acinetobacter, is almost identical to the preferred site on integrons, the composite 59-base element (59-BE) associated with this cassette is potentially recombinationally active. By using a conduction assay to quantitate site activity, the 59-BE was recognized by integrase with high frequency, indicating that the composite site is recombinationally active.

Acinetobacter↗