Inversion of Moraxella lacunata type 4 pilin gene sequences by a Neisseria gonorrhoeae site-specific recombinase

Abstract

A plasmid library of Neisseria gonorrhoeae sequences was screened for the ability to mediate recombinations on a sequence containing the Moraxella lacunata type 4 pilin gene invertible region in Escherichia coli. A plasmid containing the N. gonorrhoeae sequence encoding the putative recombinase (gcr) was identified and sequenced. Plasmids containing gcr were able to mediate site-specific recombinations despite a weak amino acid homology to Piv, the native M. lacunata pilin gene invertase. The gcr gene is present only in pathogenic strains of Neisseria tested; however, in our assays gene knockouts of gcr did not alter the variation of surface features that play a role in the pathogenesis of N. gonorrhoeae.The Piv recombinase is the only Moraxella gene product required to mediate inversion of the cloned Moraxella lacunata and Moraxella bovis type 4 pilin gene regions in Escherichia coli (25,28). The stop codons for two partial 3Ј pilin domains (tfpQ and tfpI) lie within a 2.1-kb invertible region, while the short, conserved 5Ј domain and pilin promoter lie just outside this segment (27,28). Site-specific recombinations between the boundaries of the invertible region result in transcriptional fusions of the tfpQ or tfpI domain to the conserved 5Ј pilin domain and promoter (27,31). These inversions are clearly demonstrated by use of restriction sites located asymmetrically in the invertible region and by the different mobilities of pilin subunits produced in E. coli by immunoblot analysis (31). Further highlighting the essential role for Piv in site-specific recombination, insertions into or deletions in the M. lacunata piv gene eliminate inversion of the pilin gene segment (28). Inversions of these phase-locked constructs can be restored by a plasmid carrying the piv gene (28).The type 4 pili produced by M. lacunata and M. bovis are also produced by a variety of gram-negative mammalian pathogens, including Neisseria gonorrhoeae (19,42). Pili are generally thought to play an important role in the establishment of infection by mediating the attachment to host epithelial cells and are also associated with competence for DNA transformation (4, 40), twitching motility (18), and autoagglutination (44). The gonococcal genome is well known for its ability to alter the pilin structural gene by recombination of silent pilin sequences (pilS) into the expression locus (pilE) (6,16,17,26,45). Deletions in the recA allele decrease antigenic variation of the pilin gene 100-to 1,000-fold (23), yet homologies between recombining sequences are as low as 30 bp (29), leaving open the possibility that a site-specific recombinase or other locusspecific factor facilitates the recombination process. Interestingly, there is no evidence of site-specific recombinases, phage, transposons, or insertion sequences characterized in the gonococcal genome despite this well-documented genomic plasticity. We hypothesized that if a site-specific recombinase exists in gonococci, it may be able to mediate inversions on the cloned M. lacunata type 4 pili...

Campylobacter fetusUses Multiple Loci for DNA Inversion within the 5′ Conserved Regions ofsapHomologs

“…In total, the present and previous studies (18,50) indicate that homologous recombination is the major mechanism for the high-frequency sap inversion in C. fetus and that neither the IR nor the CLS has any discernible role in this process. Studies with other bacteria have shown that the antigenic variation caused by DNA inversion may depend on the function of either RecA (31, 37, 58) or a site-specific recombinase (8,9,10,34,51). Since previous studies showed that highfrequency C. fetus sap inversion is RecA dependent (18) but that lower-frequency inversion can occur in the absence of RecA function (50), the present results are consistent with the predominant pathway.…”

Genetic Variation of the Borrelia burgdorferi Gene vlsE Involves Cassette-Specific, Segmental Gene Conversion

“…This invertase apparently recognizes specific 19-bp repeat sequences (32) at the inversion sites. A putative N. gonorrhoeae recombinase (gcr) was able to recognize the same M. lacunata sequences and to catalyze the pilin gene inversion in a surrogate Escherichia coli system, but it is still unclear whether gcr plays a role in gonococcal pilin variation or virulence (33). Gonococcal mutants deficient in pilin antigenic variation have been generated recently by transposon mutagenesis (24).…”

Bacterial insertion sequences: their genomic impact and diversity

“…A DEDD motif has also been identified in transposases of the IS110 family (Tobiason et al, 2001), closely related to the Piv and MooV invertases from Moraxella lacunata/M. bovis (Fulks et al, 1990;Rozsa et al, 1997) and N. gonorrhoeae (Choi et al, 2003;Skaar et al, 2005), respectively. Piv catalyses inversion of a DNA segment permitting expression of a type IV pilin.…”