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PubMed2004

Role of attP in integrase-mediated integration of the Shigella resistance locus pathogenicity island of Shigella flexneri.

The Shigella resistance locus (SRL) pathogenicity island (PAI) in Shigella spp. mediates resistance to streptomycin, ampicillin, chloramphenicol, and tetracycline. It can be excised from the chromosome via site-specific recombination mediated by the P4-related int gene. Here, we show that SRL PAI attP is capable of RecA-independent, site-specific, int-mediated integration into two bacterial tRNA attB sites.

Attachment Sites, Microbiological↗
PubMed1989

Characterization of the cryptic lambdoid prophage DLP12 of Escherichia coli and overlap of the DLP12 integrase gene with the tRNA gene argU.

The argU (dnaY) gene of Escherichia coli is located, in clockwise orientation, at 577.5 kilobases (kb) on the chromosome physical map. There was a cryptic prophage spanning the 2 kb immediately downstream of argU that consisted of sequences similar to the phage P22 int gene, a portion of the P22 xis gene, and portions of the exo, P, and ren genes of bacteriophage lambda. This cryptic prophage was designated DLP12, for defective lambdoid prophage at 12 min. Immediately clockwise of DLP12 was the IS3 alpha 4 beta 4 insertion element. The argU and DLP12 int genes overlapped at their 3' ends, and argU contained sequence homologous to a portion of the phage P22 attP site. Additional homologies to lambdoid phages were found in the 25 kb clockwise of argU. These included the cryptic prophage qsr' (P. J. Highton, Y. Chang, W. R. Marcotte, Jr., and C. A. Schnaitman, J. Bacteriol. 162:256-262, 1985), a sequence homologous to a portion of lambda orf-194, and an attR homolog. Inasmuch as the DLP12 att int xis exo P/ren region, the qsr' region, and homologs of orf-194 and attR were arranged in the same order and orientation as the lambdoid prophage counterparts, we propose that the designation DLP12 be applied to all these sequences. This organization of the DLP12 sequences and the presence of the argU/DLP12 int pair in several E. coli strains and closely related species suggest that DLP12 might be an ancestral lambdoid prophage. Moreover, the presence of similar sequences at the junctions of DLP12 segments and their phage counterparts suggests that a common mechanism could have transferred these DLP12 segments to more recent phages.

Amino Acid Sequence↗
PubMed1999

Interactions of the integrase protein of the conjugative transposon Tn916 with its specific DNA binding sites.

The binding of two chimeric proteins, consisting of the N-terminal or C-terminal DNA binding domain of Tn916 Int fused to maltose binding protein, to specific oligonucleotide substrates was analyzed by gel mobility shift assay. The chimeric protein with the N-terminal domain formed two complexes of different electrophoretic mobilities. The faster-moving complex, whose formation displayed no cooperativity, contained two protein monomers bound to a single DNA molecule. The slower-moving complex, whose formation involved cooperative binding (Hill coefficient > 1.0), contained four protein monomers bound to a single DNA molecule. Methylation interference experiments coupled with the analysis of protein binding to mutant oligonucleotide substrates showed that formation of the faster-moving complex containing two protein monomers required the presence of two 11-bp direct repeats (called DR2) in direct orientation. Formation of the slower-moving complex required only a single DR2 repeat. Binding of the N-terminal domains in vivo could serve to position two Int monomers on the DNA near each end of the transposon and assist in bringing together the ends of the transposon so that excision can occur. The chimeric protein with the C-terminal domain of Int also formed two complexes of different electrophoretic mobilities. The major, slower-moving complex, whose formation involved cooperative binding, contained two protein molecules bound to one DNA molecule. This finding suggested that while the C-terminal domain of Int can bind DNA as a monomer, a cooperative interaction between two monomers of the C-terminal domain may help to bring the ends of the transposon together during excision.

Base Sequence↗
PubMed2003

Characterization of the integrase gene and attachment site for the Myxococcus xanthus bacteriophage Mx9.

Bacteriophage Mx9 is a temperate phage that infects Myxococcus xanthus. It lysogenizes the bacteria by integrating into the bacterial chromosome by site-specific recombination at one of two sites, attB1 or attB2. Integration at attB1 results in deletion of DNA between the two attB sites. The attB2 site lies within the 5' region of the M. xanthus tRNA(Gly) gene. Mx9 integration requires a single protein, Int. Analysis of integration revealed that the phage attachment site (attP) is contained in the int gene and that upon integration, the 3' end of the int gene is altered. Plasmids containing fusions of the pilA or mgl promoter to lacZ integrated at either Mx9 attB site have higher levels of transcription than the same fusions integrated at the Mx8 attB site.

3' Flanking Region↗
PubMed2003

PCR typing of genetic determinants for metallo-beta-lactamases and integrases carried by gram-negative bacteria isolated in Japan, with focus on the class 3 integron.

From January 2001 to December 2002, 587 strains of gram-negative bacterial isolates demonstrating resistance to ceftazidime and a combination of sulbactam and cefoperazone were subjected to a disk diffusion screening test using sodium mercaptoacetic acid; 431 strains (73.4%) appeared to produce metallo-beta-lactamase (MBL). Of these 431 strains, 357 were found by PCR to carry genes for IMP-1 type MBL (bla(IMP-1)), while only 7 and 67 strains carried the IMP-2 gene (bla(IMP-2)) and the VIM-2 gene (bla(VIM-2)), respectively. Neither VIM-1 nor SPM-1 type MBL genes were found among the strains tested. Of 431 strains, 427 carried the intI1 gene, and 4 strains carrying both the intI1 and intI3 genes were reidentified as Pseudomonas putida harboring bla(IMP-1). Of these four P. putida strains, three strains and one strain, respectively, were separately isolated from two hospitals located in the same prefecture, and the three strains showed very similar pulsed-field gel electrophoresis patterns. Of 357 bla(IMP-1) carriers, 116, 53, 51, 47, and 30 strains were identified as Pseudomonas aeruginosa, Alcaligenes xylosoxidans, P. putida/fluorescens, Serratia marcescens, and Acinetobacter baumannii, respectively. Four strains carrying bla(IMP-2) were reidentified as P. putida. Sixty-three P. aeruginosa strains and four P. putida strains carried bla(VIM-2). Of 427 intI1-positive strains, 180, 53, 51, 47, and 35 were identified as P. aeruginosa, A. xylosoxidans, P. putida/fluorescens, S. marcescens, and A. baumannii, respectively. In the present study, it was confirmed that strains carrying bla(IMP-1) with a class 1 integron are the most prevalent type in Japan, although several intI3 carriers have also been identified sporadically in this country.

Aged↗
PubMed1991

Defining nucleic acid-binding properties of avian retrovirus integrase by deletion analysis.

Integration of retroviral DNA into the host genome requires the activity of retrovirus-encoded integration protein IN. We expressed Rous sarcoma virus (RSV) IN, 286 amino acid residues in length, by using in vitro transcription, followed by in vitro translation in rabbit reticulocyte lysate. The nucleic acid-binding activity of in vitro-translated IN was assessed by using DNA-cellulose affinity chromatography and poly(U)-Sepharose affinity chromatography and by sedimentation analysis in the presence or absence of DNA. In vitro-translated RSV IN exhibited nucleic acid-binding activity similar to that of IN purified from avian myeloblastosis virus. To identify regions of IN which bind to nucleic acids, several deletions of RSV IN were generated. The NH2-terminal 26 amino acids, including the two His residues of a His-Cys box, were not necessary for IN nucleic acid binding with any of the substrates tested. The substrates included native calf thymus DNA, poly(U), and a double-stranded linear DNA molecule with RSV long terminal repeat sequences at its termini. The COOH-terminal region (residues 178 to 286) of IN bound quantitatively (greater than 90%) to poly(U) and to single-stranded circular phi X174 DNA but did not exhibit the double-stranded linear DNA-binding ability of the entire IN molecule.

Amino Acid Sequence↗
PubMed1996

Inhibition of human immunodeficiency virus type 1 integrase by the Fab fragment of a specific monoclonal antibody suggests that different multimerization states are required for different enzymatic functions.

We have characterized a murine monoclonal antibody (MAb 35), which was raised against human immunodeficiency virus type 1 (HIV-1) integration protein (IN), and the corresponding Fab 35. Although MAb 35 does not inhibit HIV-1 IN, Fab 35 does. MAb 35 (and Fab 35) binds to an epitope in the C-terminal region of HIV-1 IN. Fab 35 inhibits 3'-end processing, strand transfer, and disintegration; however, DNA binding is not affected. The available data suggest that Fab 35 inhibits enzymatic activities of IN by interfering with the ability of IN to form multimers that are enzymatically active. This implies that the C-terminal region of HIV-1 IN participates in interactions that are essential for the multimerization of IN. Titration of the various IN-mediated enzymatic activities suggests that different degrees of multimerization are required for different activities of HIV-1 IN.

Antibodies, Monoclonal↗
PubMed1998

Implication of a central cysteine residue and the HHCC domain of Moloney murine leukemia virus integrase protein in functional multimerization.

Moloney murine leukemia virus (M-MuLV) IN-IN protein interactions important for catalysis of strand transfer and unimolecular and bimolecular disintegration reactions were investigated by using a panel of chemically modified M-MuLV IN proteins. Functional complementation of an HHCC-deleted protein (Ndelta105) by an independent HHCC domain (Cdelta232) was severely compromised by NEM modification of either subunit. Productive Ndelta105 IN-DNA interactions with a disintegration substrate lacking a long terminal repeat 5'-single-stranded tail also required complementation by a functional HHCC domain. Virus encoding the C209A M-MuLV IN mutation exhibited delayed virion production and replication kinetics.

Animals↗
PubMed2001

Moloney murine leukemia virus integrase protein augments viral DNA synthesis in infected cells.

Mutations in the IN domain of retroviral DNA may affect multiple steps of the virus life cycle, suggesting that the IN protein may have other functions in addition to its integration function. We previously reported that the human immunodeficiency virus type 1 IN protein is required for efficient viral DNA synthesis and that this function requires specific interaction with other viral components but not enzyme (integration) activity. In this report, we characterized the structure and function of the Moloney murine leukemia virus (MLV) IN protein in viral DNA synthesis. Using an MLV vector containing green fluorescent protein as a sensitive reporter for virus infection, we found that mutations in either the catalytic triad (D184A) or the HHCC motif (H61A) reduced infectivity by approximately 1,000-fold. Mutations that deleted the entire IN (DeltaIN) or 34 C-terminal amino acid residues (Delta34) were more severely defective, with infectivity levels consistently reduced by 10,000-fold. Immunoblot analysis indicated that these mutants were similar to wild-type MLV with respect to virion production and proteolytic processing of the Gag and Pol precursor proteins. Using semiquantitative PCR to analyze viral cDNA synthesis in infected cells, we found the Delta34 and DeltaIN mutants to be markedly impaired while the D184A and H61A mutants synthesized cDNA at levels similar to the wild type. The DNA synthesis defect was rescued by complementing the Delta34 and DeltaIN mutants in trans with either wild-type IN or the D184A mutant IN, provided as a Gag-IN fusion protein. However, the DNA synthesis defect of DeltaIN mutant virions could not be complemented with the Delta34 IN mutant. Taken together, these analyses strongly suggested that the MLV IN protein itself is required for efficient viral DNA synthesis and that this function may be conserved among other retroviruses.

Base Sequence↗