Mutations in simple sequence repeat tracts are a majormechanism of phase variation in several bacterial species includingCampylobacter jejuni. Changes in repeat number of tractslocated within thereading frame can produce a high frequency of reversible switches ingeneexpression between ON and OFF states. The genome of C. jejuni strainNCTC11168 contains 29 loci with polyG/polyC tracts of seven or more repeats.This protocol outlines amethod—the 28-locus-CJ11168 PV-analysis assay—forrapidly determining ON/OFF statesof 28 of these phase-variable loci in a largenumber of individual colonies from C. jejuni strainNCTC11168. The methodcombines a series of multiplex PCR assays with a fragment analysis assay andautomated extraction of fragment length, repeat number and expressionstate.This high throughput, multiplex assay has utility for detecting shifts inphase variation stateswithin and between populations over time and forexploring the effects of phase variation onadaptation to differing selectivepressures. Application of this method to analysis of the 28polyG/polyC tractsin 90 C. jejuni colonies detected a 2.5-fold increase in slippage productsastracts lengthened from G8 to G11 but no difference between tracts of similarlength indicating that flanking sequence does not influence slippage rates.Comparison of thisobserved slippage to previously measured mutation rates forG8 and G11 tracts in C. jejuniindicates that PCR amplification of a DNA samplewill over-estimate phase variation frequencies by 20-35-fold. An importantoutput of the 28-locus-CJ11168 PV-analysis assay is combinatorial expressionstates that cannot be determined by other methods. This method canbe adapted toanalysis of phase variation in other C. jejuni strains and in a diverse rangeofbacterial species.
- Campylobacter jejuni/genetics
- High-Throughput Nucleotide Sequencing/methods
- Multiplex Polymerase Chain Reaction/methods