Mutations in Haemophilus influenzae mismatch repair genes increase mutation rates of dinucleotide repeat tracts but not dinucleotide repeat-driven pilin phase variation rates

Abstract

High-frequency, reversible switches in expression of surface antigens, referred to as phase variation (PV), are characteristic of Haemophilus influenzae. PV enables this bacterial species, an obligate commensal and pathogen of the human upper respiratory tract, to adapt to changes in the host environment. Phase-variable hemagglutinating pili are expressed by many H. influenzae isolates. PV involves alterations in the number of 5' TA repeats located between the -10 and -35 promoter elements of the overlapping, divergently orientated promoters of hifA and hifBCDE, whose products mediate biosynthesis and assembly of pili. Dinucleotide repeat tracts are destabilized by mismatch repair (MMR) mutations in Escherichia coli. The influence of mutations in MMR genes of H. influenzae strain Rd on dinucleotide repeat-mediated PV rates was investigated by using reporter constructs containing 20 5' AT repeats. Mutations in mutS, mutL, and mutH elevated rates approximately 30-fold, while rates in dam and uvrD mutants were increased 14- and 3-fold, respectively. PV rates of constructs containing 10 to 12 5' AT repeats were significantly elevated in mutS mutants of H. influenzae strains Rd and Eagan. An intact hif locus was found in 14 and 12% of representative nontypeable H. influenzae isolates associated with either otitis media or carriage, respectively. Nine or more tandem 5' TA repeats were present in the promoter region. Surprisingly, inactivation of mutS in two serotype b H. influenzae strains did not alter pilin PV rates. Thus, although functionally analogous to the E. coli MMR pathway and active on dinucleotide repeat tracts, defects in H. influenzae MMR do not affect 5' TA-mediated pilin PV.

FIG. 1.

Mutational spectra of dinucleotide repeat tracts in mod-lacZ reporter genes of H. influenzae MMR mutants. Changes in repeat number for 5′ AT tracts of phase-variant colonies were classified as either insertions or deletions of particular numbers of repeat units. The numbers of variants in each class were expressed as the percentage of the total number examined. The relevant genotype, repeat tract number of parental colonies (parentheses), and number of variants examined (braces) are recorded below each column. (A) On-to-off switching. (B) Off-to-on switching in which either +1/−2 (bars 1 to 9) or −1/+2 (bars 10 to 12) reading frames produce a Mod-LacZ fusion protein. Data for mod-5′AT-lacZ reporter genes containing 20 and 22 repeats in strain Rd (wild type [wt]) and 20 and 19 repeats in RdΔmutS were published previously (5).

FIG. 2.

Analysis of the organization and promoter sequences of the hif loci of NT H. influenzae strains. (A) Representation of the hif locus of H. influenzae strain Eagan. Genes are represented by open boxes and are named. The shaded box represents the dinucleotide repeat tract. Thin arrows indicate the transcriptional orientations of the divergent hifA and hifBCDE promoters. Thick arrows indicate the primers used for PCR analysis of the hif locus. The positions of hicA and hicB were reported previously (27). (B) Alignment of the nucleotide sequences of the hif locus promoter regions from different strains. Dashes indicate nucleotides that are not present in a sequence. The transcription initiation codons (+1) and promoter elements (−10 and −35) of hifA and hifBCDE are marked by small arrowheads and dashed lines, respectively (positions are as described previously [34]). Sequence HI-AM30ed (accession number Z33502) is that of H. influenzae strain AM30 (35).