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.2005 Feb;187(4):1276-86.
doi: 10.1128/JB.187.4.1276-1286.2005.

Analysis of the Piv recombinase-related gene family of Neisseria gonorrhoeae

Affiliations

Analysis of the Piv recombinase-related gene family of Neisseria gonorrhoeae

Eric P Skaar et al. J Bacteriol.2005 Feb.

Abstract

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 invertase-related 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.

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Figures

FIG. 1.
FIG. 1.
Chromosomal location ofirg copies inN. gonorrhoeae strain FA1090. A schematic diagram of theN. gonorrhoeae chromosome is shown.pil loci are labeled, andirg copies are denoted by black arrows that point in the predicted direction of transcription. The chromosomal position of the start codon for eachirg copy is given for the linear sequence of theN. gonorrhoeae strain FA1090 genome, starting atdnaA (GenBank accession no. AE004969 [23]).
FIG. 2.
FIG. 2.
Alignment of Irg proteins with Piv and homologues. Conserved residues are boxed in gray. Asterisks are above residues shown to be required for PivML-mediated inversion ofMoraxella DNA inE. coli (40). PivNM-1A, PivNM-1B, and PivNM-2 are Piv homologues found inN. meningitidis. MooV is the transposase for IS492 inP. atlantica (21) and IS621 Tnp is the transposase for IS621 inE. coli (5).
FIG. 3.
FIG. 3.
Distribution ofirg copies in various isolates ofN. gonorrhoeae. Southern analysis was performed with chromosomal DNA preparations that were digested with ClaI, electrophoresed on a 0.8% agarose gel, transferred to a nylon membrane, and probed with denatured32P-labeled PCR product corresponding to theirg2 gene.N. gonorrhoeae strains are as follows: lane 1, FA19; lane 2, UU1; lane 3, RD5; lane 4, F62; lane 5, 1084; lane 6, 1918; lane 7, 1349; lane 8, 1402; lane 9, 1384.N. meningitidis strains are as follows: lane 10, GA0929; lane 11, NMB.Neisseria commensals are in lanes 12 (N. cineria) and 13 (N. lactamica). Negative control lanes are lanes 14 (M. lacunata) and 15 (pivML clone pAG702). The positive control,irg5 clone pAG852, is in lane 16.
FIG. 4.
FIG. 4.
Assays for complementation of Piv invertase and MooV transposase activities by Irg2 and Irg7 inE. coli. (A) Western blot analysis of His6-tagged MooV (MooV-H6), PivML (Piv-H6), Irg2 (Irg2-H6), and Irg7 (Irg7-H6) expression. Whole-cell lysates from induced (I) and uninduced (U) cultures were assayed on the Western blot as described in Materials and Methods. The proteins range in size from 318 to 322 amino acid residues without the His6 tag; the arrow marks the approximate location of the tagged Irg proteins. The molecular weights (in thousands) of protein standards (S) are indicated. (B) His6-tagged Irg7, Irg2, and the invertase PivML were assayed for invertase activity in HMS174(DE3) by using the Piv inversion substrate (pAG862), which contains theM. lacunata pilin invertible segment. Expression of the recombinases was induced and inversion products were isolated as described in Materials and Methods. Southern blot analysis of the pAG862 KpnI/SalI restriction fragments which are diagnostic for inversion of the pilin segment is shown. Arrows indicate the restriction fragments that are unique to the plasmid substrate that has undergone Piv-mediated inversion of the pilin segment, as illustrated. Tc, tetracycline. (C) His6-tagged MooV, Irg2, Irg7, and PivML were assayed for transposase activity in HMS174(DE3) by using the plasmid substrate pAG713, containing IS492ΔmooV::cat that is defective for excision due to the substitution ofcat for most of themooV transposase gene. Excision of IS492ΔmooV::cat to yield a circular element is detected in a PCR-based assay using primers (arrows) directed outward from the ends of the element which amplify the circlejunction. The 405-bp PCR product is indicated on the agarose gel by the arrow. Lane M contains pBR322/MspI (sizes in base pairs are shown). Ap, ampicillin; Kn, kanamycin.
FIG. 5.
FIG. 5.
Quantitative real-time PCR analysis ofirg transcript levels in the overexpressed strain. Transcript levels were measured by using FRET probes in a LightCycler (Roche). Black bars are levels ofirg transcripts, and gray bars are levels ofrecA transcripts included as a control. Total RNA was isolated from FA1090recA6, in whichrecA gene expression is inducible by IPTG, and FA1090recA6 NICS6::irg5, which overexpressesirg5 upon induction with IPTG.
FIG. 6.
FIG. 6.
Inactivation or overexpression ofirg genes does not affect pilin antigenic variation, DNA transformation, or DNA repair. (A) Kinetic variation assay ofN. gonorrhoeae measuring the number of sectors of a colony that undergo pilin-dependent colony variation over time. Error bars represent the standard errors of the means of at least four experiments analyzing 20 individual colonies per experiment. (B) DNA transformation competence measuring the introduction of a point mutation ingyrB that confers NAL resistance. Error bars represent the standard deviations from the means for at least four replicate experiments. (C) Relative survival after irradiation with UV light. Error bars represent the standard deviations from the means for four experiments.
FIG. 7.
FIG. 7.
Sequences flanking theirg copies and definition of ISNgo2 and ISNgo3. (A) The relative order and orientation of theirg genes are shown with flanking genes; gaps are indicated by//. Theirg1 andirg8 genes are inserted into sequences predicted to encode a conserved hypothetical protein and a nitroreductase, respectively (dark gray arrows). Putative prophage geneszot,rstA, andtspB (light gray arrows) are described in Discussion; this repeated sequence is nearly identical over 6,874 bp upstream ofirg3 andirg5, 4,836 bp downstream ofirg2, and 3,319 bp upstream ofirg6. Potential prophage sequence associated withirg7 differs from the sequence associated with otherirg genes but still encodes a filamentous phage structural protein with a Zot domain (zot2). Parentheses indicate truncated genes or sequences.hrpA is a helicase. (B) Thirty-six base pairs of sequence 5′ to the start codon and 59 bp of sequence (with a 100-bp gap of identical sequence) 3′ to the stop codon ofirg1-6 andirg8 are grouped together and aligned. Thirty-seven base pairs of sequence upstream from the start codon and 59 bp of sequence (with a 119-bp gap in which 3 bp ofirg7-associated sequence differs from those ofpivNM-1A andpivNM-1B) downstream of the stop codon ofirg7,pivNM-1A, andpivNM-1B are aligned. The completely conserved base pairs are highlighted in gray; the proposed left and right ends for ISNgo2 and ISNgo3 are boxed with a black line. Interaction of the 3′ end of theN. gonorrhoeae 16S RNA with the proposed ribosome binding sites for theirg genes of ISNgo2 and ISNgo3 is shown below the aligned sequences. dRS3 repeat sequence is underlined, and the flanking prophage sequences are italicized. (C) The putative IS elements are shown with flanking sequences to illustrate their insertion relative to the repeated prophage sequences (italicized). Theirg genes of ISNgo2 are black boxes, the left ends are white boxes, and the right ends are horizontally hatched boxes. The ISNgo3 elements, with theirg andpivNM genes as gray boxes and the left and right ends as white and diagonally hatched boxes, respectively, are inserted in the same orientation relative to prophage sequence (see above).
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