Point mutations in a peptidoglycan biosynthesis gene cause competence induction in Haemophilus influenzae

Abstract

We have identified three new Haemophilus influenzae mutations causing cells to exhibit extreme hypercompetence at all stages of growth. The mutations are in murE, which encodes the meso-diaminopimelate-adding enzyme of peptidoglycan synthesis. All are point mutations causing nonconservative amino acid substitutions, two at a poorly conserved residue (G(435)-->R and G(435)-->W) and the third at a highly conserved leucine (L(361)-->S). The mutant strains have very similar phenotypes and do not exhibit any defects in cell growth, permeability, or sensitivity to peptidoglycan antibiotics. Cells retain the normal specificity of DNA uptake for the H. influenzae uptake signal sequence. The mutations do not bypass genes known to be needed for competence induction but do dramatically increase expression of genes required for the normal pathway of DNA uptake. We conclude that the mutations do not act by increasing cell permeability but by causing induction of the normal competence pathway via a previously unsuspected signal.

FIG. 1

Time course of growth and competence of murE mutants and the wild-type parent KW20. (A) Transformation frequencies. (B) Culture densities.

FIG. 2

The murE region of the H. influenzae genome. (A) The 165-kb region around murE, showing the locations of the Tn916 insertion in strain RR783 and the Cm^r cassette insertion in strain RR797. The parentheses around the Tn916 insertion indicate that its position is only approximate. (B) The 8.0-kb region around murE cloned in plasmids p836B and p848M. Arrows indicate the direction of transcription. (C) Locations of the RR749, RR751, and RR752 point mutations (asterisks) and of the Kan^r cassette in pmurE::Kan.

FIG. 3

Comparison of murE-homologous sequences around positions 361 and 435 of the H. influenzae murE sequence. H. influ, H. influenzae; A. actino, Actinobacillus actinomycetemcomitans; Y. pestis, Yersinia pestis; P. aerug, Pseudomonas aeruginosa; N. mening, Neisseria meningitidis; B. pertus, Bordetella pertussis; C. acetob, Clostridium acetobutylicum; C. jejuni, Campylobacter jejuni; B. subtilis, Bacillus subtilis; Synecho, Synechocystis sp. strain PCC6803.

FIG. 4

Competition for DNA uptake. Strain KW20, RR563, and RR804 cells were made competent in MIV and incubated with 200 ng of MAP7 DNA/ml and the indicated concentration of competing H. influenzae or E. coli DNA. The transformation frequencies have been normalized to that seen in the absence of competing DNA.

FIG. 5

Transformation frequencies of double-mutant strains in colony assays. The error bars indicate the standard errors of the mean. The data represent one experiment (icc) or the means of two (cya, crp, rpo, rec2, dpr, and comE), three (topA and thdF), or four (KW20 and sxy) experimental points, with each point being the mean of the four colonies tested. The asterisks indicate values which are upper limits because no transformed colonies were produced by that genotype.

FIG. 6

Expression of lacZ fusions to competence genes. Solid circles, murE⁺; open circles, murE₇₄₉.