Mutations inpollbut notmutSLHdestabilizeHaemophilus influenzaetetranucleotide repeats

Abstract

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.

Involvement of genes of genome maintenance in the regulation of phase variation frequencies in Neisseria meningitidis

“…Thus, we confirm the weak mutator phenotype resulting from the inactivation of the mutS gene previously reported in N. meningitidis (Richardson & Stojiljkovic, 2001). Likewise, in H. influenzae, mutS, mutL and mutH mutants exhibit a significant three-to fourfold higher global mutation frequency than wild-type strain Rd (Bayliss et al, 2002). In addition, inactivation of dam did not result in a global mutator phenotype in N. meningitidis, in agreement with previous findings in this organism (Richardson & Stojiljkovic, 2001) and in H. influenzae strain Rd (Bayliss et al, 2002).…”

“…Likewise, in H. influenzae, mutS, mutL and mutH mutants exhibit a significant three-to fourfold higher global mutation frequency than wild-type strain Rd (Bayliss et al, 2002). In addition, inactivation of dam did not result in a global mutator phenotype in N. meningitidis, in agreement with previous findings in this organism (Richardson & Stojiljkovic, 2001) and in H. influenzae strain Rd (Bayliss et al, 2002). Strain MC58 does not appear to have the mutH gene , suggesting that mismatch repair in N. meningitidis may be different from the system characterized in E. coli.…”

“…Dam methylation was shown to have no effect upon phase variation of tetranucleotides in H. influenzae (Bayliss et al, 2002) and of polyG tracts in N. meningitidis (Richardson & Stojiljkovic, 2001). In contrast, Bucci et al (1999) proposed that the absence of Dam activity was responsible for the high frequency of N. meningitidis capsule phase variation and for the heightened virulence of some strains.…”

“…In Escherichia coli, the overexpression of dinB, which encodes the error-prone DNA polymerase IV, results in an increased frequency of frameshifts in short homopolymeric tracts (Kim et al, 1997). In Haemophilus influenzae, inactivation of mutS, a key component of MMR, and polI destabilize dinucleotide and tetranucleotide repeat tracts, respectively (Bayliss et al, 2002). In N. meningitidis, investigation of two contingency loci revealed that the mismatch repair genes mutS and mutL have a role in controlling phase variation frequencies (Richardson & Stojiljkovic, 2001;Richardson et al, 2002).…”

Hypermutable Haemophilus influenzae with mutations in mutS are found in cystic fibrosis sputum

“…The mutation rates to rifampicin and nalidixic acid resistance of strain Rd KW20 mutS : : TSTE (R3544) are about eightfold greater than the mutation frequency of the parent strain Rd KW20 (R652) due to insertional inactivation of the MMR protein mutS (Watson et al, 2004). These mutation rates are comparable to previously reported mutation rates to rifampicin and nalidixic acid resistance for strains Rd KW20 and Rd KW20 DmutS (Bayliss et al, 2002(Bayliss et al, , 2004. The mutation rates of the natural hypermutable isolates were variable and ranged from about 5-to 380-fold greater than Rd KW20 (R652).…”