Deletion analysis of the subcloned DNA inversion region of MoraxeUla lacunata indicates that Piv is the only M. lacunata-encoded factor required for site-specific inversion of the tlpQIip'I pilin segment. The predicted amino acid sequence of Piv shows significant homology solely with the transposases/integrases of a family of insertion sequence elements, suggesting that Piv is a novel site-specific recombinase.Moraxella lacunata and Moraxella bovis are human and bovine ocular pathogens, respectively (4,17,27). Both of these gram-negative pathogens produce type 4 (MePhe) pili, which are important virulence factors mediating adherence of the bacilli to corneal tissue (7,22,23,29). M. bovis and M. lacunata utilize site-specific DNA inversion of a chromosomal segment to alter the expression of their type 4 pilin genes (22, 28). M. bovis alternately expresses Q and I pilin, and M. lacunata apparently exhibits an on/off pilin phase variation (23, 30). The 2.1-kb invertible DNA segment in M. bovis contains the carboxyl-terminal portion of the tfpQ and tfpI pilin genes and an additional open reading frame, ORF1 or tfpB ( Fig. 1) (21).The promoter and constant region of the pilin genes is located upstream of the invertible segment. The DNA inversion region of M. lacunata is very similar in sequence and organization to that of M. bovis, with the notable exceptions of a 19-bp duplication within the coding sequence for the carboxylterminal region of the tfpI pilin gene, resulting in no active pilin expression in one orientation of the invertible segment, and a 2-bp deletion in tfpB which shortens the ORF by one-third (28).A subcloned 6-kb fragment containing the inversion region of M. lacunata in a pBluescript vector (pMxLl; Fig. 1 (22), has the inversion region from M. lacunata (same region encoded on pMxLl), but piv has been inactivated by insertion of a segment from the fl interposon encoding streptomycin/spectinomycin resistance. The deletion of tfpB was accomplished by restriction of pMxL5 with AfllI and SnaBI (New England Biolabs) (Fig. 1) and ligation of the annealed oligonucleotides 5'-TTAAGATCGATGACGTCAGATCTG AGCTCGATACTCGAG-3' and 5'-CTCGAGTATCGAGCT CAGATCT'GACGTCATCGATC-3' into the Aflll and SnaBI sites, using T4 DNA ligase (Boehringer) to create pAG701. Inversion assays were then performed by transforming E. coli DH5a containing pMxL5 or pAG701 with a compatible plasmid, pACYCpiv-1 (provided by Meredith Hackel and Carl Marrs), which is pACYC184 containing the M. bovis piv gene under control of its own promoter. The transformants and DH5a strains containing pMxL5 or pAG701 were grown in culture overnight selecting for chloramphenicol (pACYC piv-1) and/or ampicillin (pMxL5/pAG701). Plasmid DNA was then isolated and digested with KpnI, which cuts once asymmetrically within the invertible segment and once in the pBluescript vector DNA (Fig. 1); electrophoresis of restriction fragments was done as described by Marrs et al. (22). Digestion of pMxL5 with KpnI in the absence of Piv results in fragments of 9.4 and 0.5 k...
Neisseria gonorrhoeae (the gonococcus) is an obligate human pathogen and the causative agent of the disease gonorrhea. The gonococcal pilus undergoes antigenic variation through high-frequency recombination events between unexpressed pilS silent copies and the pilin expression locus pilE. The machinery involved in pilin antigenic variation identified to date is composed primarily of genes involved in homologous recombination. However, a number of characteristics of antigenic variation suggest that one or more recombinases, in addition to the homologous recombination machinery, may be involved in mediating sequence changes at pilE. Previous work has identified several genes in the gonococcus with significant identity to the pilin inversion gene (piv) from Moraxella species and transposases of the IS110 family of insertion elements. These genes were candidates for a recombinase system involved in pilin antigenic variation. We have named these genes irg for invertaserelated gene family. In this work, we characterize these genes and demonstrate that the irg genes do not complement for Moraxella lacunata Piv invertase or IS492 MooV transposase activities. Moreover, by inactivation of all eight gene copies and overexpression of one gene copy, we conclusively show that these recombinases are not involved in gonococcal pilin variation, DNA transformation, or DNA repair. We propose that the irg genes encode transposases for two different IS110-related elements given the names ISNgo2 and ISNgo3. ISNgo2 is located at multiple loci on the chromosome of N. gonorrhoeae, and ISNgo3 is found in single and duplicate copies in the N. gonorrhoeae and Neisseria meningitidis genomes, respectively.
The recombinase, Piv, is essential for site-specific DNA inversion of the type IV pilin DNA segment in Moraxella lacunata and Moraxella bovis. Piv shows significant homology with the transposases of the IS110/ IS492 family of insertion elements, but, surprisingly, Piv contains none of the conserved amino acid motifs of the Int or Hin/Res families of site-specific recombinases. Therefore, Piv may mediate site-specific recombination by a novel mechanism. To begin to determine how Piv may assemble a synaptic nucleoprotein structure for DNA cleavage and strand exchange, we have characterized the interaction of Piv with the DNA inversion region of M. lacunata. Gel shift and nuclease/chemical protection assays, competition and dissociation rate analyses, and cooperativity studies indicate that Piv binds two distinct recognition sequences. One recognition sequence, found at multiple sites within and outside of the invertible segment, is bound by Piv protomers with high affinity. The second recognition sequence is located at the recombination cross-over sites at the ends of the invertible element; Piv interacts with this sequence as an oligomer with apparent low affinity. A model is proposed for the role of the different Piv binding sites of the M. lacunata inversion region in the formation of an active synaptosome.Numerous site-specific DNA recombination systems and DNA transposition systems have been characterized biochemically and have been found to follow two distinct chemical pathways for DNA cleavage and strand transfer in recombination (reviewed in Refs. 1-4). Site-specific recombination, mediated by the recombinases of the -integrase and Hin/resolvase families, involves a two-step transesterification reaction in which the intermediate is a covalent recombinase-DNA linkage. This covalent attachment is the result of nucleophilic attack on the DNA phosphodiester backbone by a hydroxyl group of the conserved serine (Hin/resolvase), or tyrosine (-integrase), of the recombinase. In the second transesterification reaction, the phosphodiester linkages of the exchanged DNA strands are restored (reviewed in Refs. 2 and 3). In contrast, DNA transposition, mediated by transposases containing the catalytic DDE amino acid motif, utilizes a hydrolysis reaction for cleavage at the ends of the transposable element. This first cleavage leaves 3Ј-OH ends to act directly as the attacking nucleophile in a one-step trans-esterification reaction resulting in strand exchange. Resolution of the transposition process involves DNA replication or DNA repair activity to fill in gaps left at the target site due to the staggered cut mediated by the transposase and the 3Ј hydroxyl groups at the element ends (reviewed in Refs. 1 and 4).These features of the recombination reactions mediated by site-specific recombinases and transposases suggest that a group of related recombinases would not mediate both sitespecific recombination and transposition. Therefore, it is surprising that the site-specific recombinase Piv, which directs site-specific DN...
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