Experimentally revised repertoire of putative contingency loci in Neisseria meningitidis strain MC58: evidence for a novel mechanism of phase variation

Analysis of the genome sequence of Neisseria meningitidis strain MC58 revealed 65 genes associated with simple sequence repeats. Experimental evidence of phase variation exists for only 14 of these 65 putatively phase variable genes. We investigated the phase variable potential of the remaining 51 genes. The repeat tract associated with 20 of these 51 genes was sequenced in 26 genetically distinct strains. This analysis provided circumstantial evidence for or against the phase variability of the candidate genes, based on the sequence and the length of the repeated motif. These predictions of phase variability were substantiated for three of these candidate genes using colony immunoblotting or beta-galactosidase as a reporter. This investigation identified a novel phase variable gene (NMB1994 or nadA) associated with a repeat tract (TAAA) not previously reported to be associated with phase variable genes in N. meningitidis. Analysis of the nadA transcript revealed that the repeat tract was located upstream of the putative -35 element of the nadA promoter. Semiquantitative RT-PCR showed that variation in the number of repeats was associated with changes in the level of expression of nadA, findings consistent with a model whereby the variable number of (TAAA) repeats modulates the promoter strength.