SummaryHaemophilus in¯uenzae is an obligate commensal of the upper respiratory tract of humans that uses simple repeats (microsatellites) to alter gene expression. The mod gene of H. in¯uenzae strain Rd has homology to DNA methyltransferases of type III restriction/modi®cation systems and has 40 tetranucleotide (58-AGTC) repeats within its open reading frame. This gene was found in 21 out of 23 genetically distinct H. in¯uenzae strains, and in 13 of these strains the locus contained repeats. H. in¯uenzae strains were constructed in which a lacZ reporter was fused to a chromosomal copy of mod downstream of the repeats. Phase variation occurred at a high frequency in strains with the wild-type number of repeats. Mutation rates were derived for similarly engineered strains, containing different numbers of repeats. Rates increased linearly with tract length over the range 17±38 repeat units. The majority of tract alterations were insertions or deletions of one repeat unit with a 2:1 bias towards contractions of the tract. These results demonstrate the number of repeats to be an important determinant of phase variation rate in H. in¯uenzae for a gene containing a microsatellite.

SummaryAnalysis 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 b b b b -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.

Haemophilus influenzae (Hi), an obligate upper respiratory tract commensal/pathogen, uses phase variation (PV) to adapt to host environment changes. Switching occurs by slippage of nucleotide repeats (microsatellites) within genes coding for virulence molecules. Most such microsatellites in Hi are tetranucleotide repeats, but an exception is the dinucleotide repeats in the pilin locus. To investigate the effects on PV rates of mutations in genes for mismatch repair (MMR), insertion/deletion mutations of mutS, mutL, mutH, dam, polI, uvrD, mfd and recA were constructed in Hi strain Rd. Only inactivation of polI destabilized tetranucleotide (5'AGTC) repeat tracts of chromosomally located reporter constructs, whereas inactivation of mutS, but not polI, destabilized dinucleotide (5'AT) repeats. Deletions of repeats were predominant in polI mutants, which we propose are due to end-joining occurring without DNA polymerization during polI-deficient Okazaki fragment processing. The high prevalence of tetranucleotides mediating PV is an exceptional feature of the Hi genome. The refractoriness to MMR of hypermutation in Hi tetranucleotides facilitates adaptive switching without the deleterious increase in global mutation rates that accompanies a mutator genotype.