A genetic screen identifies genes and sites involved in pilin antigenic variation in Neisseria gonorrhoeae

Abstract

SummaryIt has previously been shown that the frequency of pilin antigenic variation in Neisseria gonorrhoeae (the gonococcus, Gc) is regulated by iron availability. To identify factors involved in pilin variation in an irondependent or an iron-independent manner, we conducted a genetic screen of transposon-mutated gonococci using a pilus-dependent colony morphology phenotype to detect antigenic variation deficient mutants. Forty-six total mutants representing insertions in 30 different genes were shown to have reduced colony morphology changes resulting from impaired pilin variation. Five mutants exhibited an iron-dependent decrease in pilin variation, while the remaining 41 displayed an iron-independent decrease in pilin variation. Based on the levels of antigenic variation impairment, we defined the genes as being essential for, important for, or involved in antigenic variation. DNA repair and DNA transformation frequencies of each mutant were measured to determine whether other recombination-based processes were also affected in the mutants. Each mutant was placed into one of six classes based on their pilin variation, DNA repair and DNA transformation phenotypes. Among the many genes identified, recR is shown to be an additional member of the gonococcal RecF-like recombination pathway. In addition, recG and ruvA represent the first evidence that the processing of Holliday junctions is required for pilin antigenic variation. Moreover, two independent insertions in a noncoding region upstream of the pilE gene suggest that cis -acting sequences important for pilin variation are found in that region. Finally, insertions that effect expression of the thrB and thrC genes suggest that molecules in the threonine biosynthetic pathway are important for pilin variation. Many of the other genes identified in this genetic screen do not have an obvious role in pilin variation, DNA repair, or DNA transformation.

The Gonococcal NlpD Protein Facilitates Cell Separation by Activating Peptidoglycan Cleavage by AmiC

“…We insertionally inactivated the nlpD gene by inserting a cat resistance gene cassette in place of the coding sequence. The strain FA1090 nv (the subscript "nv" in the strain name indicates that a strain cannot undergo pilin antigenic variation [24]) used in this study contained either 1-81-S2 or RM11.2 pilE variant sequences. Each of the FA1090 variants were transformed with the mutant ⌬nlpD::cat allele construct (here designated ⌬nlpD) by spot transformation, and Cam r colonies were selected on Cam plates.…”

Mutation of the priA Gene of Neisseria gonorrhoeae Affects DNA Transformation and DNA Repair

“…During pilin antigenic variation, a variable portion of a pilS copy replaces the corresponding portion of pilE, recombining at short regions of identity shared between the silent and expressed genes (16). Pilin antigenic variation is dependent on recA (21), the recF-like pathway (33,57), rdgC (32), and rep (19) as well as the ruvABC and recG branch migration pathways (50).…”

“…Recombinational repair is well studied in N. gonorrhoeae and requires the recA (21) and recX (62) genes, along with either the RecBCD pathway components recB, recC, and recD (33) or the RecF pathway components recO, recR, recQ, and recJ (33,50,57). The branch migration genes recG and ruvA also contribute to recombinational DNA repair in N. gonorrhoeae (50). The use of multiple genetic pathways for recombinational DNA repair in N. gonorrhoeae distinguishes it from Escherichia coli, which utilizes the RecF DNA repair pathway only in a mutant recBC sbcBC background (22,25).…”

DprA is required for natural transformation and affects pilin variation in Neisseria gonorrhoeae

“…Pilin variation was measured using the previously described pilus-dependent colony morphology changes (PDCMC) assay (Sechman et al, 2005), which monitors formation of faster-growing P À colony outgrowths that emerge on the edge of P + colonies over time with the following modifications. P + and P À colonies vary morphologically and are indicative of pilus expression.…”

“…To test this, we measured pilin variation of the dprA mutants using a colony morphology proxy for pilin variation. PDCMC, which are indicative of pilin variation (Sechman et al, 2005), were measured and quantified in our strains. Cells lacking functional DprA exhibited significantly higher pilin variation compared to parental cells in both FA1090 and MS11 (P<0.05, Fig.…”